EA045605B1 - TARGETTING LIGANDS FOR THERAPEUTIC COMPOUNDS - Google Patents

Description

Cross reference to related applications

This application claims priority to the U.S. Provisional Patent Application Serial No.

62/304652, filed March 7, 2016, and US Provisional Patent Application No. 62/370754, filed August 4, 2016, and US Provisional Patent Application No. 62/426916, filed November 28, 2016, contents of each which are incorporated herein by reference in their entirety.

State of the art

To obtain a therapeutic effect or be useful for diagnostic purposes, many compounds must be delivered to specific sites (eg, target cell(s)). This is often the case when attempting to deliver a therapeutic compound in vivo. In addition, the ability to efficiently deliver a compound to a specific site may limit or largely eliminate off-target effects (such as side effects) that may be caused by administration of the compound. One method of achieving delivery of a compound, such as a therapeutic compound, to a desired site in vivo is based on binding or coupling of the compound to a targeting ligand.

One class of therapeutic compounds that can be targeted using targeting ligands are oligomeric compounds. Oligomeric compounds that include nucleotide sequences at least partially complementary to the target nucleotide sequence have been shown to alter the function and activity of the target both in vitro and in vivo. When delivered into a cell containing a target nucleotide sequence (such as mRNA), the oligomeric compounds have been shown to modulate the expression of the target, resulting in changes in the transcription or translation of the target nucleic acid. In some cases, the oligomeric compound may reduce gene expression by inhibiting the target nucleic acid and/or triggering the destruction of the target nucleic acid.

If the target nucleic acid is mRNA, one mechanism by which an expression inhibitory oligomeric compound can modulate the expression of the target mRNA is through RNA interference. RNA interference is a biological process that allows RNA or RNA-like molecules (such as chemically modified RNA molecules) to silence gene expression (silence) by destruction. Post-translational gene silencing is thought to be an evolutionarily conserved cell defense mechanism to prevent the expression of foreign genes.

Synthetic RNA molecules and RNA-like molecules have been shown to cause RNA interference in vivo. For example, Elbashir et al. (Nature 2000, 411, 494-98) describe RNAi performed by introducing duplexes of synthetic 21-nucleotide RNA molecules into cultured mammalian cells. The types of synthetic RNA molecules and RNA-like molecules that can trigger an RNAi response may include modified nucleotides and/or one or more linkages other than a phosphodiester linkage.

Additionally, single-stranded RNA molecules and RNA-like molecules, which may also include modified nucleotides and/or one or more linkages other than a phosphodiester linkage, can also alter the expression of a target nucleic acid, such as target mRNA.

Short description

Disclosed herein are targeting ligands that can enhance the delivery of therapeutic compounds to a specific site, for example, a specific organ or tissue in a subject, such as a human or animal patient. In some embodiments, the targeting ligands described herein can improve the targeting of expression inhibitory oligomeric compounds. In some embodiments, targeting ligands can improve delivery of expression inhibitory oligomeric compounds to the liver.

The targeting ligands disclosed herein include or consist of one or more targeting moieties, one or more tethers, one or more branch point-containing groups, and one or more linkers.

Disclosed herein are targeting ligands that include, consist of, or consist essentially of the general structure of Formula A

where n is an integer from 1 to 4 (for example, 1, 2, 3 or 4).

In some embodiments, the targeting ligands disclosed herein include, consist of, or consist essentially of the general structure of Formula B:

- 1 045605

TARGETING FRAGMENT

TARGETING FRAGMENT

TARGETING FRAGMENT

where n is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20);

X is O, S or NH and the targeting moiety is selected from the group consisting of N-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, N-h-butanoylgalactosamine and N-isobutanoylgalactosamine.

In some embodiments, the targeting ligands disclosed herein include, consist of, or consist essentially of the following structure:

where n is an integer from 1 to 20 (Structure 1).

In some embodiments, the disclosed targeting ligands include, consist of, or consist essentially of a structure selected from

The targeting ligands disclosed herein include one or more targeting moieties. In some embodiments, the targeting ligands disclosed herein include N-acetylgalactosamine as a targeting moiety.

The targeting ligands disclosed herein can be coupled directly or indirectly to a compound, such as a therapeutic compound, such as an expression inhibitory oligomeric compound, for example, to the 3' or 5' end of the expression inhibitory oligomeric compound. In some embodiments, the expression inhibitory oligomeric compound includes one or more modified nucleotides. In some embodiments, the expression inhibitory oligomeric compound is an RNAi agent, such as a double-stranded RNAi agent. In some embodiments, the targeting ligands disclosed herein are coupled to the 5' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligands disclosed herein are linked to the RNAi agent by a phosphate, phosphorothioate, or phosphonate group at the 5' end of the sense strand of the double-stranded RNAi agent.

Disclosed herein are compositions containing a targeting ligand and an expression inhibitory oligomeric compound. Disclosed herein are compositions containing a targeting ligand and an RNAi agent.

In some embodiments, compositions disclosed herein containing a targeting ligand and an RNAi agent have the structure represented by where Z includes or consists of an expression inhibitory oligomeric compound (Structure 101a);

where Z includes or consists of an expression inhibitory oligomeric compound (Structure 102a); and where Z includes or consists of an expression inhibitory oligomeric compound (Structure 103a).

Disclosed herein are phosphoramidite compounds comprising targeting ligands.

In some embodiments, the phosphoramidite compounds comprising the targeting ligands disclosed herein have the structure represented by where n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

- 3 045605

15, 16, 17, 18, 19 or 20) (Structure 1d);

(Structure 101d);

(Structure 103 d).

Also disclosed are pharmaceutical compositions that include the targeting ligands disclosed herein.

Disclosed are methods of treating a disease or disorder that would benefit from administration of a therapeutic oligomeric compound, comprising administering to a subject a therapeutic oligomeric compound linked to a targeting ligand disclosed herein.

Disclosed herein are methods of inhibiting the expression of a target nucleic acid in a subject, comprising administering a therapeutic amount of an expression inhibitory oligomeric compound coupled to targeting ligands disclosed herein.

Disclosed herein are methods of delivering an expression inhibitory oligomeric compound to the liver in vivo, comprising administering the expression inhibitory oligomeric compound bound to a targeting ligand disclosed herein to a subject.

As used herein, the term bonded, when applied to a connection between two molecules, means that the two molecules are connected by a covalent bond or that the two molecules are connected by non-covalent bonds (eg, hydrogen bonds or ionic bonds). In some examples where the term bound refers to the association of two molecules by non-covalent bonds, this association (association) of two different molecules is characterized by a KD below 1x10'4 M (for example, below ^1x10'5 M, below 1x10'6 M, or below 1x10 ^-7 M) in a physiologically acceptable buffer (for example, phosphate buffer solution).

As used herein, the term bound directly refers to a first compound or group associated with a second compound or group without any intervening atoms or groups of atoms. As used herein, the term indirectly linked refers to a first compound linked to a second compound or group via an intermediate group, compound or molecule, such as, for example, a linker group. Unless otherwise specified, the term related herein includes both directly related and indirectly related as those terms are defined herein.

As used herein, an oligomeric compound is a nucleotide sequence containing about 10-50 nucleotides or nucleotide base pairs. In some embodiments, the oligomeric compound has a sequence of nucleotide bases that

- 4 045605 is at least partially complementary to a coding sequence in the expressed target nucleic acid or target gene in the cell. In some embodiments, the oligomeric compounds, once delivered to a cell expressing a gene, are capable of inhibiting the expression of the corresponding gene and are referred to herein as expression-inhibitory oligomeric compounds. Gene expression can be inhibited in vitro or in vivo. Oligomeric compounds include, but are not limited to: oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (kuRNA, siRNA), double-stranded RNA (dsRNA, lncRNA), microRNA (miRNA, microRNA), short hairpin RNA (shRNA, shRNA ), ribozymes, molecules mediating RNA interference and dicer sebsstrates.

As used herein, the term oligonucleotide refers to a polymer of linked nucleosides, each of which may be modified or unmodified.

As used herein, the term single-stranded oligonucleotide refers to a single-stranded oligomeric compound having a sequence that is at least partially complementary to a target mRNA, which is capable of hybridizing to the target mRNA by hydrogen bonding under normal physiological conditions in a mammal (or under comparable in vitro conditions). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

As used herein, an RNAi Agent refers to an agent that contains an RNA or RNA-like oligonucleotide molecule (e.g., a chemically modified RNA oligonucleotide) that is capable of disrupting or inhibiting the translation of messenger RNA transcripts (mRNA) of target mRNA in a sequence-specific manner. As used herein, RNAi agents may act by RNA interference (i.e., causing RNA interference by interacting with components of the RNA interference pathway (RNA-induced gene silencing complex or RISC) of mammalian cells), or by account of any alternative mechanism(s) or path(s). Although RNAi agents, as that term is used herein, are believed to act primarily through the mechanism of RNA interference, the disclosed RNAi agents are not associated with or limited to any particular pathway or mechanism of action. RNAi agents include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (khRNA, siRNA), double-stranded RNA (dsRNA, lncRNA), microRNA (miRNA, microRNA), short hairpin RNA (shRNA), and dicersebstrates. The RNAi agents described herein consist of an oligonucleotide whose strand is at least partially complementary to the mRNA being targeted. In some embodiments, the RNAi agents described herein are double-stranded, consisting of an antisense strand and a sense strand that is at least partially complementary to the sense strand. RNAi agents may include modified nucleotides and/or one or more linkages other than a phosphodiester linkage. In some embodiments, the RNAi agents described herein are single-stranded.

As used herein, the terms silence, reduce, inhibit, suppress, or knockdown, as applied to the expression of a given gene, mean that the expression of that gene, as measured by the level of RNA transcribed from the gene, or the level of polypeptide, protein, or protein subunit translated from that gene The mRNA in a cell, group of cells, tissue, organ or body of a subject in which a given gene is transcribed is reduced when that cell, group of cells, organ or subject is treated with oligomeric compounds bound to the targeting ligands described herein compared to a second a cell, group of cells, tissue, organ or subject that has not been subjected to such treatment.

As used herein, the term sequence or nucleotide sequence refers to a sequence or order of nucleotide bases or nucleotides described by a sequence of letters using standard nomenclature for nucleotides.

As used herein and unless otherwise indicated, the term complementary to the description of a first nucleotide sequence (e.g., an RNAi Agent sense strand or target mRNA) with respect to a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded RNAi Agent antisense strand), denotes the ability of the oligonucleotide or a polynucleotide comprising said first nucleotide sequence, hybridize (form hydrogen bonds in a base pair under physiological conditions of a mammalian body (or under comparable in vitro conditions)) and form a duplex or double helix structure under specified conditions with an oligonucleotide or polynucleotide comprising a second nucleotide subsequence. Complementary sequences include Watson-Crick and non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimetics, at least as long as the hybridizability requirements above are met.

As used herein, absolutely complementary or fully complementary means that all (100%) of the nucleotides in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide. The contiguous sequence may include all or part of the first or second nucleotide sequence.

As used herein, partially complementary means that in a hybridized pair of nucleotide base sequences, at least 70%, but not all, of the bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide.

As used herein, substantially complementary means that in a hybridized pair of nucleotide base sequences, at least 85%, but not all, of the bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide. The terms complementary, fully complementary, and substantially complementary as used herein may be used to refer to a match between a sense strand and an antisense strand of a double-stranded RNAi agent, between an antisense strand of a double-stranded RNAi agent and a target mRNA sequence, or between a single-stranded antisense oligonucleotide sequence and a target mRNA sequence.

As used herein, the terms treat, cure, and the like refer to methods and measures taken to ensure a reduction in the number, severity and/or frequency of one or more symptoms of a disease in a subject.

As used herein, the phrase cellular administration, when referring to an oligomeric compound, means the functional delivery of the oligomeric compound into a cell. The phrase functional delivery means the process of delivering an oligomeric compound into a cell in a manner that allows the oligomeric compound to exhibit the intended biological activity, for example, sequence-specific inhibition of gene expression.

Unless otherwise indicated, the use of the symbol Ά herein means that any group or groups that are within the scope of the compound disclosed herein may be associated with it.

As used herein, the term isomers refers to compounds that have the same molecular formula but differ in the nature or sequence of bonds between atoms or the arrangement of atoms in space. Isomers that differ in the arrangement of atoms in space are called stereoisomers. Stereoisomers that are not mirror images of each other are called diastereoisomers, and stereoisomers that are incompatible mirror images are called enantiomers, or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is called a chiral center.

In this text, unless an asymmetric center is specifically stated to have a structure in a particular conformation, for each structure in which asymmetric centers are present and can give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to encompass mixtures of diastereomers as well as individual stereoisomers.

The term substituted (containing substituents) as used herein means that any one or more hydrogens on the designated atom, usually a carbon, oxygen or nitrogen atom, is replaced by any group designated herein, provided that the normal valency of the designated atom is not exceeded. atom, and that this substitution produces a stable compound. Non-limiting examples of substituents include C1- _C6 alkyl, _C2 - _C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, keto, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen ( for example, F, Cl, Br, I). If the substituent is keto or oxo (i.e., =O), then two (2) hydrogens on the indicated atom are replaced. Ring double bonds as used herein are double bonds formed between two adjacent atoms on the ring (eg, C=C, C=N, N=N, etc.).

Some of the compounds described herein may exist in a tautomeric form, which is also included within the scope of this description. Tautomers are compounds whose structures differ significantly in the arrangement of atoms, but which exist in an easily and quickly established equilibrium. It should be understood that the compounds described herein may be represented as various tautomers. It should also be understood that where compounds have tautomeric forms, all tautomeric forms are intended to be included within the scope of this specification, and the names assigned to the compounds do not exclude any tautomeric form.

The compounds and pharmaceutically acceptable salts disclosed herein may exist in one or more tautomeric forms, including ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid on heterocyelic rings (e.g., the nitrogenous bases guanine,

- 6 045605 thymine and cytosine), amine - enamine and enamine - enamine, as well as in the form of geometric isomers and their mixtures. Ring tautomerism, exhibited by glucose and other sugars, results from the interaction of an aldehyde group (-CHO) on a sugar molecule with one of the hydroxy groups (-OH) on the same molecule, resulting in the formation of a cyclic (ring) form. All such tautomeric forms are included within the scope of the present invention. Tautomers exist as mixtures of a group of tautomers in solution. Even though a single tautomer may be described, the present invention includes all tautomers of the compound disclosed herein. The concept of tautomers that can change into each other through tautomerization is called tautomerism. In tautomerism, a simultaneous shift of electrons and a hydrogen atom occurs.

Various types of tautomerization are catalyzed by:

Reason:

1) deprotonation;

2) formation of a delocalized anion (for example, enolate);

3) protonation at various positions of the anion;

Acid:

1) protonation;

2) formation of a delocalized cation;

3) deprotonation at various positions adjacent to the cation.

As used herein, the term alkyl refers to a saturated aliphatic hydrocarbon group, linear or branched, containing from 1 to 10 carbon atoms, unless otherwise noted. For example, C1-Cb alkyl includes alkyl groups containing 1, 2, 3, 4, 5 or 6 carbon atoms in a linear or branched configuration. As used herein, the term aminoalkyl refers to an alkyl group, as defined above, substituted at any position by one or more amino groups as permitted by the rules of valency. Amino groups can be unsubstituted, monosubstituted or disubstituted.

As used herein, the term cycloalkyl refers to a saturated or unsaturated non-aromatic hydrocarbon ring group containing from 3 to 14 carbon atoms, unless otherwise indicated. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl or cyclohexyl, etc. Cycloalkyls may contain multiple spiro or fused rings. Cycloalkyl groups are optionally mono-, di-, tri-, tetra- or penta-substituted at any position, as long as valence rules allow this.

As used herein, the term alkenyl refers to a non-aromatic hydrocarbon radical, which may be linear or branched, containing at least one carbon-carbon double bond and from 2 to 10 carbon atoms unless otherwise specified. Such groups can contain up to five carbon-carbon double bonds. For example, C ₂ -C ₆ is defined as an alkenyl radical containing from 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl and cyclohexenyl. The linear, branched or cyclic moiety of an alkenyl group may contain double bonds and is optionally mono-, di-, tri-, tetra- or penta-substituted at any position as permitted by normal valence. The term cycloalkenyl refers to a monocyclic hydrocarbon group containing the specified number of carbon atoms and at least one carbon-carbon double bond.

As used herein, the term alkynyl refers to a carbohydrate radical, linear or branched, containing from 2 to 10 carbon atoms, unless specifically stated otherwise, and at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be present. Accordingly, C ₂ -C ₆ alkynyl denotes an alkynyl radical containing from 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to: ethynyl, 2propynyl and 2-butynyl. The linear or branched portion of an alkynyl group may contain triple bonds as permitted by conventional valency requirements, and may optionally be mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by conventional valency.

As used herein, alkoxy or alkoxy refers to an alkyl group as defined above, with the number of carbon atoms indicated, attached by an oxygen bridge. _C1-6 alkoxy is meant to include C1, _C2 , _C3 , _C4 , _C5 and _C6 alkoxy groups. _{C1_8} alkoxy is meant to include C1, _C2 , _C3 , _C4 , _C5 , _C6 , C7 and _C8 alkoxy groups. Examples of alkoxy include, but are not limited to: methoxy, ethoxy, n-propoxy, n-propoxy, n-butoxy, sec-butoxy, t-butoxy, n-pentoxy, secpentoxy, n-heptoxy and n-octoxy.

As used herein, keto refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl or aryl group as defined herein, attached by a carbonyl bridge. Examples of a keto group include, but are not limited to: alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, hexanoyl), aclenoyl (e.g., acryloyl), alkinoyl (e.g., ethinoyl, propinoyl, butinoyl, pentinoyl, hexanoyl), aryloyl ( e.g. benzoyl), heteroaryloyl (e.g. pyrroloyl, imidazoyl, quinoli

- 7 045605 noyl, pyridinoyl).

As used herein, alkoxycarbonyl refers to any alkoxy group as defined above attached by a carbonyl bridge (ie, -C(O)O-alkyl). Examples of alkoxycarbonyl groups include, but are not limited to: methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or npentoxycarbonyl.

As used herein, aryloxycarbonyl refers to any aryl group as defined herein attached via an oxycarbonyl bridge (ie, -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to: phenoxycarbonyl and naphthyloxycarbonyl.

As used herein, heteroaryloxycarbonyl refers to any heteroaryl group as defined herein attached via an oxycarbonyl bridge (ie, -C(O)heteroaryl). Examples of heteroaryloxycarbonyl groups include, but are not limited to: 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

As used herein, aryl or aromatic means any stable monocyclic or polycyclic carbohydrate ring containing up to 7 atoms on each ring, at least one ring being aromatic. Examples of aryl groups include, but are not limited to: phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl and biphenyl. In the case where the aryl substituent is bicyclic and one ring is non-aromatic, the addition is assumed to occur via an aromatic ring. Aryl groups are not necessarily mono-, di-, tri-, tetra- or penta-substituted in any position as permitted by normal valency.

As used herein, the term heteroaryl represents a stable monocyclic or polycyclic ring containing up to 7 atoms on each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Examples of heteroaryl groups include, but are not limited to, the following: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolinyl, imidazopyridinyl, isoindolinyl, indazolyl, oxa zolyl, oxadiazolyl , isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinolinyl. Heteroaryl is also intended to include the N-oxide derivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain heteroatoms, the addition is assumed to occur through an aromatic ring or a ring containing a heteroatom. Heteroaryl groups are not necessarily mono-, di-, tri-, tetra- or penta-substituted in any position as permitted by normal valency.

As used herein, the term heterocycle, heterocyclic or heterocyclyl refers to a 314-membered aromatic or non-aromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, including polycyclic groups. As used herein, the term heterocyclic is also considered synonymous with the terms heterocycle and heterocyclyl and is intended to be as defined herein. Heterocyclyl includes the above-mentioned heteroaryls, as well as their dihydro- and tetrahydro-analogues. Examples of heterocyclyl groups include, but are not limited to, the following: azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinols l , isothiazolyl, isoxazolyl, naftpyridinyl, oxadiazolyl, oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl , pyrimidyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl , quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepenyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrho lidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl ,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl and tetrahydrothienyl and their N-oxides. The heterocyclyl substituent can be attached via a carbon atom or via a heteroatom. Heterocyclyl groups are not necessarily mono-, di-, tri-, tetra- or penta-substituted in any position as permitted by normal valency.

One of ordinary skill in the art will readily understand and appreciate that the compounds and compositions disclosed herein may contain certain atoms (e.g., N, O, or S atoms) in the

- 8 045605 toned or deprotonated state, depending on the environment in which this compound or composition is placed. Accordingly, the structures disclosed herein provide that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. The present disclosure is intended to cover the disclosed compounds and compositions regardless of their protonation status as determined by the pH of the environment; one skilled in the art will readily understand this.

As used herein, the expression consisting of excludes any elements, steps or ingredients not specified in the claim. When used in the claims of this document, the expression consisting essentially of limits the scope of the claim to specified materials or steps and materials or steps that do not significantly affect the essential and novel feature or characteristics of the claimed invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning usually given to them by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein may be used to practice or study the present invention, suitable methods and materials are described below. The contents of all publications, patent applications, patents and other references noted herein are incorporated herein by reference in their entirety. In case of conflict, the present description, including definitions, controls. Moreover, the materials, methods, and examples are for illustrative purposes only and are not limiting.

Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief description of drawings

Fig. 1 is the 1H NMR spectrum of compound 3 (as described below in Example 1).

Fig. 2 is the 1H NMR spectrum of compound 4 (as described below in Example 1).

Fig. 3 is the 1H NMR spectrum of compound 6 (as described below in Example 1)

Fig. 4 is the 1H NMR spectrum of compound 7 (as described below in Example 1)

Fig. 5 is the 1H NMR spectrum of compound 9 (as described below in Example 1).

Fig. 6 is the 1H NMR spectrum of compound 10 (which is Structure 101d herein and described below in Example 1).

Fig. 7 is the 1H NMR spectrum of compound 13 (which is Structure 103d herein and described below in Example 2).

Fig. 8 is the 1H NMR spectrum of compound 16 (which is Structure 102d herein and described below in Example 3).

Fig. 9 is an HPLC chromatogram for AM03704 conjugated to Structure 103d (as described below in Example 5).

Fig. 10 is an HPLC chromatogram for AM03704 conjugated to Structure 101d (as described below in Example 5).

Fig. 11 is an HPLC chromatogram for AM03704 conjugated to Structure 102d (as described below in Example 5).

Fig. 12 is a graph illustrating normalized levels of mouse Factor 12 (F12) protein in wild-type mice (as described below in Example 6).

Fig. 13 is a graph illustrating normalized levels of mouse Factor 12 (F12) protein in wild-type mice (as described below in Example 7).

Fig. 14 is a graph illustrating normalized levels of lipoprotein(a) particles (Lp(a)) in Lp(a) Tg transgenic mice (as described below in Example 8).

Fig. 15 is a graph illustrating normalized levels of lipoprotein(a) particles (Lp(a)) in Lp(a) Tg transgenic mice (as described below in Example 9).

Fig. 16 is a graph illustrating normalized apo(a) levels in apo(a) transgenic (Tg) mice (as described below in Example 10).

Fig. 17 is a graph illustrating normalized cF12 protein levels in cynomolgus monkeys (as described below in Example 12).

Fig. 18 is a graph illustrating normalized AAT protein levels (Z-AAT) in PiZ transgenic mungweeds (as described below in Example 13).

Fig. 19 is a graph illustrating normalized levels of mouse Factor 12 (F12) protein in wild-type mice (as described below in Example 14).

Detailed description

New targeting ligands are described that are associated with compounds such as therapeutic or diagnostic expression inhibitory oligomeric compounds. In some embodiments, the compounds that are associated with the targeting ligands described herein include or consist of therapeutic compounds that are RNAi agents. Targeting ligands can be used to target therapeutic compounds to the desired position of a target nucleic acid or gene. Also disclosed herein are compositions including targeting ligands and therapeutic compounds, such as compositions including or consisting of targeting ligands and expression-inhibiting oligomeric compounds.

The novel targeting ligands disclosed herein provide efficient targeting or biodistribution, sufficient in vivo and in vitro stability, and can be synthesized as phosphoramidites, which reduces the cost and complexity of manufacture, and may improve the potency relative to previously discussed inhibitory targeting ligands. expression by an oligomeric compound such as an RNAi agent.

Targeting ligands

Targeting ligands consist of one or more targeting group(s) or targeting moiety(s) that can serve to improve the pharmacokinetics and biodistribution properties of the compound to which they are bound and improve the cell- or tissue-specific distribution and cell-specific uptake of the conjugated composition. In general, a targeting ligand facilitates the targeted delivery of the therapeutic compound to which it is bound to the desired target site. In some cases, the targeting moiety may bind to a cell or cell receptor and initiate endocytosis, facilitating entry of the therapeutic compound into the cell. The targeting moiety may include compounds having affinity for cellular receptors or cell surface molecules or antibodies. Various targeting ligands, which contain targeting moieties, can be associated with therapeutic agents and other compounds to target the agents to cells and specific cellular receptors. Types of targeting moieties include carbohydrates, cholesterol and cholesterol groups, and steroids. Targeting moieties that can bind to cellular receptors include saccharides such as galactose, galactose derivatives (such as N-acetylgalactosamine), mannose and mannose derivatives; other carbohydrates; glycans; haptens, vitamins, folate; biotin, aptamers; and peptides such as RGD-containing peptides, insulin, EGF and transferrin.

Targeting moieties known to bind to the asialoglycoprotein receptor (ASGPR) are particularly useful for directing the delivery of oligomeric compounds to the liver. Asialoglycoprotein receptors are expressed in large quantities on liver cells, including hepatocytes. Targeting moieties for cellular receptors that target ASGPR include galactose and galactose derivatives. In particular, clusters of galactose derivatives, including clusters consisting of two, three or four N-acetylgalactosamines (GalNAc or NAG), may facilitate the uptake of certain compounds into liver cells. GalNAc clusters conjugated to oligomeric compounds act to direct the composition to the liver, where N-acetylgalactosamine sugars can bind to asialoglycoprotein receptors on the surface of liver cells. Binding to the asialoglycoprotein receptor is thought to trigger receptor-mediated endocytosis, thereby facilitating entry of the compound into the cell.

The targeting ligands disclosed herein may include one, two, three, four, or more than four targeting moieties. In some embodiments, the targeting ligands disclosed herein may contain one, two, three, four, or more than four targeting moieties linked to a branch point group. In some embodiments, the targeting ligands disclosed herein may comprise one, two, three, four, or more than four targeting moieties linked to a branch point group, with each targeting moiety linked to the branch point group by a connecting moiety.

In some embodiments, the targeting ligands disclosed herein may comprise one, two, three, four, or more than four asialoglycoprotein receptor (ASGPR) targeting moieties linked to a branch point group. In some embodiments, the targeting ligands disclosed herein may comprise ASGPR) linked to a branch point group, with each targeting moiety for the ASGPR linked to the branch point group by a connecting moiety.

The targeting ligands described herein are represented by Formula I below

where n is an integer from 1 to 4 (for example, 1, 2, 3 or 4) (Formula I). In some embodiments, n in Formula I is an integer from 1-3, 1-2, 2-4, 2-3, or 3-4.

The targeting ligands disclosed herein can be coupled to therapeutic compounds, such as oligomeric compounds. In some embodiments, the targeting ligand is linked to the therapeutic compound by an additional linker and/or cleavable

- 10 045605 fragment, which in turn is associated with a therapeutic compound. In some embodiments, the targeting ligands are ligated (linked) to the therapeutic compound itself.

In some embodiments, the therapeutic compound is an expression inhibitory oligomeric compound. In some embodiments, the expression inhibitory oligomeric compound is an RNAi agent. In some embodiments, the expression inhibitory oligomeric compound is a double-stranded RNAi agent.

In some embodiments, the targeting ligand is connected, directly or indirectly, to the 5' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligand is connected, directly or indirectly, to the 3' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligand is connected, directly or indirectly, to the 5' end or 3' end of the antisense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligand is connected, directly or indirectly, to the 5' end or 3' end of the single-stranded RNAi agent.

In some embodiments, the targeting ligand is linked to the double-stranded RNAi agent through a phosphate, phosphonate, phosphorothioate, or internucleoside linking group at the 5' end of the terminal nucleoside of the sense strand of the double-stranded RNAi agent.

In some embodiments, the targeting ligand disclosed herein includes a cleavable moiety. In some embodiments, the cleavable moiety includes or consists of a phosphate or other internucleoside linking group that can be cleaved. In some embodiments, the targeting ligand is linked to the therapeutic compound by a cleavable moiety.

In some embodiments, the targeting ligand disclosed herein is linked to additional group or groups that include a cleavable moiety. In some embodiments, the targeting ligand is linked to a cleavable moiety, which in turn is linked to an expression inhibitory oligomeric compound.

In some embodiments, the binding ligand is a phosphoramidite compound (also referred to herein as a phosphoramidite-containing compound). The phosphoramidite compound comprising the targeting ligand described herein can be used to readily attach the targeting ligand to the therapeutic compound or other moieties using phosphoramidite synthesis methods well known in the art. In some embodiments, the phosphoramidite compound comprising the binding ligand is coupled to the expression inhibitory oligomeric compound using methods well known in the art. In some embodiments, the targeting ligand-containing phosphoramidite is linked to the 5' end of the sense strand of the double-stranded RNAi agent.

In some embodiments, the expression inhibitory oligomeric compound associated with the targeting ligand includes a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide. In some embodiments, the targeting ligand is linked directly to the single-stranded antisense oligonucleotide. In some embodiments, additional groups are inserted between the targeting ligand and the single-stranded oligonucleotide.

In some embodiments, the targeting ligand associated with the RNAi agent includes one or more N-acetylgalactosamine sugars as a targeting moiety or targeting moieties.

In some embodiments, the targeting ligand associated with the expression inhibitory oligomeric compound includes a connecting moiety that includes polyethylene glycol (PEG). In some embodiments, the connecting moiety consists of PEG. In some embodiments, the connecting moiety includes a PEG containing from 1 to 10 ethylene glycol units. In some embodiments, the connecting moiety includes a PEG containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol units.

In some embodiments, the targeting ligand associated with the RNAi agent contains polyethylene glycol (PEG) as a linker. In some embodiments, the linker comprises PEG. In some embodiments, the linker consists of PEG. In some embodiments, the linker comprises a PEG containing from 1 to 20 ethylene glycol units. In some embodiments, the connecting moiety comprises a PEG containing from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene glycol units.

In some embodiments, the expression inhibitory oligomeric compound associated with any of the targeting ligands disclosed herein includes an RNAi agent. In some embodiments, the targeting ligand disclosed herein is linked, directly or indirectly, to an RNAi agent.

In some embodiments, the targeting ligand disclosed herein is linked directly to the RNAi agent. In some embodiments, the targeting ligand disclosed herein is linked indirectly to the RNAi agent because an additional group or groups are embedded between the RNAi agent and the targeting ligand linker. In some embodiments, a second linker is included between the linker and the therapeutic compound.

Structures of targeting ligands and phosphoramidite compounds incorporating targeting ligands.

The targeting ligands disclosed herein may consist of one or more targeting moieties, tethers, branch point-containing groups, and linkers. The targeting ligands disclosed herein may contain one, two, three, four, or more than four targeting moieties.

In some embodiments, the targeting ligands disclosed herein are synthesized such that they are in the form of a phosphoramidite compound. Phosphoramidites are widely used in the chemical synthesis of RNA and DNA. In some embodiments, phosphoramidite-containing targeting ligands disclosed herein are coupled to the 5' end of the sense strand of a double-stranded RNAi agent. It may be particularly useful to provide the targeting ligand in the form of a phosphoamidite if the targeting ligand is coupled to the 5' end of the expression inhibitory oligomeric compound. Without intending to be limited by theory, it is believed that providing the targeting ligand as a phosphoramidite, where the targeting ligand is linked to the 5' end of an expression inhibitory oligomeric compound, not only allows the targeting ligand to be attached as a final component (which reduces manufacturing cost), but also potentially allows targeting ligand to block the sense strand from entering RISC if the targeting ligand is attached to the 5' end of the sense strand of a double-stranded RNAi agent. If the expression inhibitory oligomeric compound is a double-stranded RNAi agent, the targeting ligand can be provided in the form of a phosphoramidite compound, wherein the targeting ligand is coupled to the 5' end of the sense strand of the RNAi agent.

In some embodiments, the targeting ligand is represented by Formula B:

where n is an integer from 1 to 20;

X is O, S or NH and the targeting moiety is selected from the group consisting of galactose, galactosamine, Nformylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine or N-iso-butanoylgalactosamine (Formula B). In some embodiments, n is 6. In some embodiments, n is 8. In some embodiments, n is 4.

In some embodiments, the targeting ligand has the structure shown below

where n is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) (Structure 1).

In some embodiments, the targeting ligand has the structure represented by Structure 1, where n=6. In some embodiments, the targeting ligand has the structure represented by Structure 1, where n=8. In some embodiments, the targeting ligand has the structure represented by Structure 1, where n=4.

In some embodiments, the binding ligand is linked to an expression inhibitory oligomeric compound and has the structure shown below

- 12 045605

where Z includes or consists of an expression inhibitory oligomeric compound (Structure 1a).

In some embodiments, the binding ligand is linked to an expression inhibitory oligomer and has the structure shown below:

where Z consists of or includes an expression inhibitory oligomeric compound (Structure 1b). In some embodiments, the binding ligand is linked to an expression inhibitory oligomeric compound and has the structure shown below

where Z consists of or includes an expression inhibitory oligomeric compound (Structure 1c).

In some embodiments, the binding ligand is a phosphoramidite containing compound having the structure shown below:

where n is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) (Structure 1d).

In some embodiments, the targeting ligand contains or consists of a structure shown as follows:

In some embodiments, the binding ligand is linked to an expression inhibitory oligomer and has the structure shown below

- 13 045605

where Z includes or consists of an expression inhibitory oligomeric compound (Structure 101a).

In some embodiments, the binding ligand is linked to an expression inhibitory oligomeric compound and has the structure shown below

where Z consists of or includes an expression inhibitory oligomeric compound (Structure 101b).

In some embodiments, the binding ligand is linked to an expression inhibitory oligomeric compound and has the structure shown below

where Z consists of or includes an expression inhibitory oligomeric compound (Structure 101c).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure set forth below

In some embodiments, the targeting ligand contains or consists of a structure shown as follows:

In some embodiments, the binding ligand is linked to an expression inhibitory oligomeric compound and has the structure shown below: it

where Z includes or consists of an expression inhibitory oligomeric compound (Structure 102a).

In some embodiments, the binding ligand is linked to an expression inhibitory oligomer and has the structure shown below:

where Z consists of or includes an expression-inhibiting oligomeric compound (Structure 102b).

In some embodiments, the binding ligand is linked to an expression inhibitory oligomeric compound and has the structure shown below:

where Z consists of or includes an expression inhibitory oligomeric compound (Structure 102c).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

(Structure 102d).

In some embodiments, the targeting ligand comprises or consists of a structure represented by:

- 15 045605

In some embodiments, the binding ligand is linked to an expression inhibitory oligomer and has the structure shown below

where Z includes or consists of an expression inhibitory oligomeric compound (Structure 103a).

In some embodiments, the binding ligand is linked to an expression inhibitory oligomer and has the structure shown below

where Z consists of or includes an expression inhibitory oligomeric compound (Structure 103b).

In some embodiments, the binding ligand is linked to an expression inhibitory oligomeric compound and has the structure shown below

where Z consists of or includes an expression inhibitory oligomeric compound (Structure 103c).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure set forth below

(Structure 103 d).

In some embodiments, the targeting ligand has the structure shown below

- 16045605

(Structure 2)

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

He

where Z consists of or includes an expression inhibitory oligomeric compound;

A represents O or S and

A' is O', S' or NH' (Structure 2b).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure set forth below

and o (Structure 2d).

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is associated with a targeting ligand and has the structure shown below: it

where Z consists of or includes an expression inhibitory oligomeric compound;

A represents O or S and

A' represents O', S' or NH' (Structure 3b).

In some embodiments, the binding ligand is a phosphoramidite containing compound having the structure set forth below

- 17045605

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where Z consists of or includes an expression inhibitory oligomeric compound;

A represents O or S and

A' is O ^- , S ^- or NH ^- (Structure 4b).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure set forth below

In some embodiments, the targeting ligand has the structure shown below

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below

- 18 045605

where Z consists of or includes an expression inhibitory oligomeric compound;

A represents O or S and

A' is O ^- , S ^- or NH ^- (Structure 5b).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure set forth below

In some embodiments, the targeting ligand has the structure shown below

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below

where Z consists of or includes an expression inhibitory oligomeric compound;

A represents O or S and

A' is O ^- , S ^- or NH ^- (Structure 6b).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure set forth below

In some embodiments, the targeting ligand is in the form of a galactose cluster. IN

- 19 045605 herein, the galactose cluster includes a targeting ligand containing two to four galactose derivatives at the end. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. The galactose derivative is a saccharide sugar, which is a type of targeting moiety. The terminal galactose derivative can be linked to the connecting moiety via the C-1 carbon of the saccharide.

In some embodiments, the targeting ligand consists of three terminal galactosamines or galactosamine derivatives (such as N-acetylgalactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand includes three terminal N-acetylgalactosamines (GalNAc or NAG) as targeting moieties.

In some embodiments, the targeting ligand consists of four terminal galactosamines or galactosamine derivatives (such as N-acetylgalactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand includes four terminal N-acetylgalactosamines (GalNAc or NAG) as targeting moieties.

In some embodiments, each targeting moiety includes a galactosamine derivative that is N-acetylgalactosamine. Other saccharides having affinity for the asialoglycoprotein receptor that can be used as targeting moieties can be selected from the list including galactose, galactosamine, Nformylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine and N-isobutanoylgalactosamine. The affinities of numerous galactose derivatives for the asialoglycoprotein receptor have been studied (see, for example: Iobst, S.T., Drickamer, K.J.B.C. 1996, 277, 6686) or can be easily determined using methods well known and widely used in the art.

Terms commonly applied in the art to the three terminal N-acetylgalactosamines include triantennary, trivalent and trimeric (trimer).

Linkers

The targeting ligands disclosed herein contain a linker.

A linker is a group of atoms linked to a branch point group at one end (or to a phosphoramidite phosphoramidite atom via a phosphotylation reaction with a phosphoramidite-forming reagent if the targeting ligand is synthesized as a phosphoramidite compound) at the other end. In some embodiments, the linker is coupled to a branch point group at one end, and ligated at the other end to a group or groups that are further ligated to an expression inhibitory oligomeric compound. In some embodiments, the linker is directly linked to the oligomeric compound. In some embodiments, the linker is associated with a cleavable moiety, which in turn is associated with an oligomeric compound. Examples of cleavable moieties include, for example, phosphate groups, groups including a disulfide moiety, and/or other internucleotide linkages that can be cleaved. In some embodiments, the linker is not associated with the cleavable moiety. In some embodiments, the linker is linked to a phosphorothioate or phosphonate group.

In some embodiments, the linker consists of or includes a polyethylene glycol (PEG) moiety. Incorporation of a PEG moiety into a linker imparts certain advantageous properties relative to other linkers, such as linkers that consist of or include substituted or unsubstituted alkyl chains. For example, inclusion of a PEG moiety in the linker increases the solubility of the targeting ligand-containing phosphoramidite compound in solvents commonly used in nucleotide synthesis compared to compounds that contain alkyl chain linkers, which may provide simplified manufacturing methods.

In some embodiments, the targeting ligand comprises a linker having the following structure:

where n is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) (Structure 1001).

In some embodiments, the targeting ligand comprises a linker attached to a phosphate group having the following structure:

where n is an integer selected from 1 to 20 (Frame 1002).

In some embodiments, the targeting ligand comprises a linker linked to a phosphorothioate group having the following structure:

- 20 045605 s * where n is an integer selected from numbers from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19 or 20) (Structure 1003).

In some embodiments, the targeting ligand comprises a linker having the following structure:

⁶ /(Structure 1004).

In some embodiments, the targeting ligand comprises a linker attached to a phosphate group having the following structure:

° ^? (Structure 1005).

In some embodiments, the targeting ligand comprises a linker attached to a phosphorothioate group having the following structure: o o s * (Structure 1006).

In some embodiments, the linker is coupled to an expression inhibitory oligomeric compound that is a double-stranded RNAi agent. In some embodiments, the linker is linked to the 5' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the linker is linked to the 3' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the linker is linked to the 3' end of the antisense strand of the double-stranded RNAi agent. In some embodiments, the linker is linked to the 5' end of the antisense strand of the double-stranded RNAi agent.

In some embodiments, the linker is associated with a cleavable moiety. In some embodiments, the terminal phosphate group of the expression inhibitory oligomeric compound may serve as a cleavable moiety. In some embodiments, an independently selected cleavable moiety is coupled to a linker. As used herein, a cleavable fragment is a group that is stable outside the cell but is cleaved once it enters the target cell. Cleavable fragments are cleaved under certain conditions, such as pH, or in the presence of certain cleavage agents, such as molecules that stimulate degradation or redox agents.

In some embodiments, the cleavable moiety may be pH sensitive. For example, it is known that endosomes and lysosomes in general have a more acidic pH (pH approximately 4.5 to 6.5) than human blood (pH approximately 7.35 to 7.45) and thus stimulate degradation of the cleaved fragment.

In some embodiments, the cleavable moiety is a phosphate group. Phosphate groups can be cleaved by agents known to resolve or hydrolyze phosphate groups.

Branch point groups

The targeting ligands disclosed herein contain at least one branch point group. The branch point group of the targeting ligands disclosed herein is attached to a linker. In some embodiments, the branch point moiety of the targeting ligands disclosed herein is coupled to a linker at one end, and the branch point moiety is coupled to one or more connecting moieties at the other end(s). In some embodiments, the branch point group is attached to a linker and one or more connecting moieties. In some embodiments, the branch point group is linked indirectly (eg, by a linker) to the expression inhibitory oligomeric compound. In some embodiments, the branch point group is linked to the expression inhibitory oligomeric compound via an additional group or groups.

Branch point groups disclosed herein can be any group that is capable of being attached to one or more targeting moieties and is further capable of being attached to a linker.

The branch point groups disclosed herein can be any group that allows the attachment of two, three or four galactose derivatives and further allows the branch point to be connected to a linker.

In some embodiments, the targeting ligand comprises a branch point having the following structures:

- 21 045605

Tethers

The targeting ligands disclosed herein contain one or more additional moieties. The connecting moiety is positioned as a link between the branch point group and each targeting moiety. In some embodiments, the connecting moiety is linked directly to a targeting ligand at one end and directly to a branch point group at the other end. In some embodiments, the connecting moiety is linked directly to a targeting ligand at one end, and indirectly to a branch point group at the other end. In some embodiments, the connecting moiety is linked indirectly to a targeting ligand at one end and indirectly to a branch point group at the other end. In some embodiments, the targeting ligand described herein includes three connecting moieties and three targeting moieties. In some embodiments, the targeting ligand described herein includes four connecting moieties and four targeting moieties. In some embodiments, a targeting ligand described herein includes one connecting moiety and one targeting moiety. In some embodiments, a targeting ligand described herein includes a plurality of connecting moieties and a plurality of targeting moieties.

In some embodiments, additional connecting moieties and other groups are embedded between the connecting moiety and the targeting moiety. In some embodiments, a second connecting moiety is embedded between the connecting moiety and the targeting moiety. In some embodiments, the second connecting fragment and the third connecting fragment are embedded between the connecting fragment and the targeting fragment. In some embodiments, the second, third, and fourth connecting moieties are embedded between the connecting moiety and the targeting moiety. As disclosed herein, there is at least one connecting moiety per targeting moiety. In some embodiments, there is more than one connecting moiety per targeting moiety. The targeting ligands disclosed herein are intended to cover all such compositions.

In some embodiments, additional groups may be embedded between the connector portion and the branch point group.

As disclosed herein, the connecting moiety serves as a spacer that can further increase the flexibility and/or length of the bond between the targeting moiety and the branch point group, linker, and therapeutic compound. In some embodiments, the connecting moiety includes alkyl groups (including cycloalkyl groups), alkenyl groups (including cycloalkenyl groups), alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, or aralkynyl groups. In some embodiments, the connecting moiety includes one or more heteroatoms, heterocycles, heteroaryls, amino acids, nucleotides, or saccharides.

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

where n is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) and

X is O, S or NH (Structure 301).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

where X is O, S or NH (Structure 302).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

- 22 045605

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

where n is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) and

X is O, S or NH (Structure 303).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

where n is an integer from 1 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) and

X is O, S or NH (Structure 304).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

(Structure 305).

where X represents O, S or NH.

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

(Structure 306).

where X represents O, S or NH.

In some embodiments, the targeting ligand includes more than one type of connecting moieties. In some embodiments, the connecting moiety acts as a flexible hydrophilic spacer (see, for example, US 5885968; and Biessen et al. J. Med. Chem. 1995, 39, 15381546, both of which are incorporated herein by reference in their entirety) and includes PEG spacer. In other embodiments, the PEG spacer contains from 1 to 20 ethylene units (PEG1PESH ₂₀ ). For example, a PEG spacer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ethylene units.

Targeting fragments:

The targeting ligands disclosed herein may contain from one to four, or more than four targeting moieties.

In some embodiments, the targeting ligand may be a galactose cluster. As used herein, the galactose cluster includes a targeting ligand containing two to four galactose derivatives at the end. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. The galactose derivative is a saccharide sugar, which is a type of targeting moiety. The terminal galactose derivative is linked to the connecting moiety through the C-1 carbon of the saccharide.

In some embodiments, the targeting ligand consists of three terminal galactosamines or galactosamine derivatives (such as N-acetylgalactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand includes three terminal N-acetylgalactosamines (GalNAc or NAG) as targeting moieties. For example, Structures 1, 101, 102 and 103 are each targeting ligands containing three terminal N-acetylgalactosamines as targeting moieties.

In some embodiments, each targeting moiety includes a galactosamine derivative that is N-acetylgalactosamine. Other saccharides having affinity for the asialoglycoprotein receptor that can be used as targeting moieties can be selected from the list including: galactose, galactosamine, Nformylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine and N-isobutanoylgalactosamine. The affinities of numerous galactose derivatives for the asialoglycoprotein receptor have been studied (see, e.g., Iobst, S.T., Drickamer, K.J.B.C. 1996, 271, 6686, which is incorporated herein by reference in its entirety) or can be readily determined using methods well known and widely used in this area.

- 23 045605

In some embodiments, the targeting moiety is a cell targeting moiety.

In some embodiments, the targeting moiety includes N-acetylgalactosamine:

In some embodiments, the targeting ligand includes three targeting moieties. In some embodiments, the targeting ligand includes four targeting moieties. In some embodiments, the targeting ligand includes a single targeting moiety. In some embodiments, the targeting ligand includes two targeting moieties. In some embodiments, the targeting ligand includes four or more targeting moieties.

In some embodiments, the targeting moiety includes one or more of galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-nbutanoylgalactosamine, or N-isobutanoylgalactosamine.

For example, in some embodiments, the N-acetylgalactosamine targeting moieties in any of Structures 1 through 6 may be replaced by alternative targeting moieties. In some embodiments, the N-acetylgalactosamine targeting moieties in any of Structures 101, 102, or 103 may be replaced by alternative targeting moieties. Such alternative targeting moieties include, for example, galactose, galactosamine, Nformylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, or N-isobutanoylgalactosamine.

Additionally, in some embodiments, the targeting moieties in Structures 1 to 6 may be replaced, for example, by other carbohydrates; glycans; haptens, vitamins, folate; biotin, aptamers; and/or peptides such as RGD-containing peptides, insulin, EGF and/or transferrin. In some embodiments, the targeting moieties in Structures 101, 102 or 103 may be replaced, for example, by other carbohydrates; glycans; haptens, vitamins, folate; biotin, aptamers; and/or peptides such as RGD-containing peptides, insulin, EGF and/or transferrin.

In some embodiments, the targeting ligand is in the form of a N-acetylgalactosamine trimer. In some embodiments, the targeting ligand is in the form of an N-acetylgalactosamine tetramer.

Oligomeric compounds

The targeting ligands disclosed herein may be associated with an oligomeric compound. In some embodiments, the oligomeric compound is an expression inhibitory oligomeric compound. In some embodiments, the expression inhibitory oligomeric compound is an RNAi agent. In some embodiments, the expression inhibitory oligomeric compound is a double-stranded RNAi agent. In some embodiments, the expression inhibitory oligomeric compound is a single-stranded oligonucleotide. Expression inhibitory oligomeric compounds can be synthesized using methods commonly used in the art.

The expression inhibitory oligomeric compounds may include one or more modified nucleotides. A nucleotide base (or nitrogenous base) is a heterocyclic pyrimidine or purine compound that is a constituent component of all nucleic acids and includes adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). As used herein, the term nucleotide may include a modified nucleotide or nucleotide mimetic, a region without a nitrogenous base, or a surrogate replacement fragment. As used herein, a modified nucleotide is a nucleotide, a nucleotide mimetic, a site without a nitrogenous base, or a surrogate replacement fragment other than a ribonucleotide (2'hydroxyl nucleotide). In some embodiments, the modified nucleotide includes a 2' modified nucleotide (i.e., a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring). Modified nucleotides include, but are not limited to: 2'-modified nucleotides, 2'-O-methylnucleotides (represented herein by the lowercase 'n' in the nucleotide sequence), 2'-deoxy-2'fluoronucleotides (represented herein as Nf, also referred to herein as 2'-fluoronucleotide), 2'-deoxynucleotides (represented herein as dN), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, (represented herein as NM or 2'-MOE), 2'-aminonucleotides, 2'-alkylnucleotides, 3'-3' linkage (inverted) nucleotides (represented herein as invdN, invN, invn, invX), non-natural bases, including closed nucleotides nucleotides, bridging nucleotides, peptide nucleic acids, 2',3' seconucleotide mimetics (uncapped nucleobase analogues, referred to herein as Nuna or NUNA), closed nucleotides (represented herein as Nlna or NLNA), 3' -O-methoxy (with an internucleotide linkage at the 2' position) nucleotide (represented herein as 3'-OMen), 2'H arabinonucleotides (represented herein as NfANA or Nf _ANA ), morpholinonucleotides, vinyl phosphonate deoxyribonucleotide (represented herein herein as vpdN), vinylphosphonate nucleotides, and nucleotide without a nucleoside base (represented herein as X or Ab). It is not necessary that all positions of a given connection be modified in the same way. Conversely, more than one modification may be made to a single expression inhibitory oligomeric compound or even to a single oligonucleotide thereof. Expression inhibitory oligomeric compounds can be synthesized and/or modified by methods known in the art. The modification of each nucleotide is independent of the modifications of other nucleotides.

Modified nucleobases include synthetic and natural nucleobases such as 5-substituted pyrimidines, 6-azapyrimidines, N-2-, N-6-, and O-6-substituted purines (e.g., 2-aminopropyladenine), 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, containing 6-methyl and other alkyl derivatives of adenine and guanine, containing 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil , 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothimine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo- , 8-amino-, 8-thiol-, 8-thioalkyl-, 8-hydroxyl- and other 8-substituted adenines and guanines, 5-substituted uracils and cytosines (for example, 5-halo-uracils and cytosines (for example, 5-bromouracil and 5- bromocytosine), 5-trifluoromethyl uracil, 5-trifluoromethyl cytosine), 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

For the expression inhibitory oligomeric compounds described herein, any modified nucleotides may be linked by phosphate-containing or phosphate-free covalent bonds. Modified internucleoside linkages or scaffolds include, but are not limited to, the following: 5'-phosphorothioate group (referred to herein as a lowercase 's' before the nucleotide, as in sN, sn, sNf or sdN), chiral phosphorothioates, thiophosphate, phosphorodithioates, phosphotriesters , aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidate and aminoalkylphosphoramidates, thiopophosphoramidates, thionoalkylphosphonates, thiopoalkylphosphotriesters, morpholino bonds, borano phosphates containing the usual 3'-5'- bonds, 2'-5' linked analogues of boranophosphonates and boranophosphonates with inverted polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, the modified internucleotide linkage or scaffold lacks a phosphorus atom. Modified internucleotide linkages without a phosphorus atom include, but are not limited to: short-chain alkyl or cycloalkyl sugar linkages, mixed heteroatom-sugar and alkyl or cycloalkyl linkages, or one or more short-chain heteroatom or cycloalkyl sugar linkages . In some embodiments, modified internucleoside frameworks include, but are not limited to: siloxane frameworks, sulfide frameworks, sulfoxide frameworks, sulfone frameworks, formacetyl and thioformacetyl frameworks, methyleneformacetyl and thioformacetyl frameworks, alkene-containing frameworks, sulfamate frameworks, methylene imine and methylene hydrazine frameworks, sulfonate and sulfonamide frameworks, amide frameworks, and other frameworks containing a mixture of N, O, S and CH2 components.

In some embodiments, the expression inhibitory oligomeric compound is a double-stranded RNAi agent and includes a sense strand and an antisense strand that are at least partially complementary (at least 70% complementary) to each other. The antisense strand contains a region having a sequence that is completely complementary (100% complementary) or at least substantially complementary (at least 85% complementary) to the sequence of the target mRNA. The length of each of the sense and antisense strands of a double-stranded RNAi agent can range from 16 to 30 nucleotides in length. The sense and antisense strands can be the same length or different lengths. In some embodiments, the sense strand is about 19 nucleotides long and the sense strand is about 21 nucleotides long. In some embodiments, the sense strand is about 21 nucleotides in length and the sense strand is about 23 nucleotides in length. In other embodiments, the length of each of the sense and antisense strands is independently 17-21 nucleotides. In some embodiments, both the sense and antisense strands are 21-26 nucleotides in length. In some embodiments, both the sense and antisense strands are 26 nucleotides in length. In some embodiments, the length of each of the sense and antisense strands is independently from 17 to 26 nucleotides. In some embodiments, the double-stranded RNAi agent comprises a duplex of about 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. The length of the region of complete or substantially complete complementarity between the sense strand and the antisense strand is

- 25 045605 is usually 15-25 (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides) and is located at or near the 5' end of the antisense strand.

Expression inhibitory oligomeric compounds that are conjugated to the ligands disclosed herein optionally and additionally include 1, 2, 3, 4, 5, or 6 additional nucleotides (as an extension) at the 3' end, 5' end, or both. 3' and 5' ends of the main sequences. These additional nucleotides, if present, may or may not be complementary to the corresponding sequence in the target mRNA.

In some embodiments, when a double-stranded RNAi agent is conjugated to targeting ligands disclosed herein, the additional sense strand nucleotides, if present, may or may not be identical to the corresponding sequence in the target mRNA. Additional antisense strand nucleotides, if present, may be complementary or non-complementary to corresponding additional sense strand nucleotides, if present.

Double-stranded RNAi agents can be formed by combining an antisense strand with a sense strand.

In some embodiments, the targeting ligand is linked to the RNAi agent at the 3' or 5' end of either the sense or antisense strand of the RNAi agent. In some embodiments, the targeting ligand is associated with the 5' end of the sense strand. In some embodiments, the targeting ligand is associated with the 3' end of the sense strand. In some embodiments, the targeting ligand is linked to the RNAi agent by a labile, cleavable, or reversible bond. In some embodiments, the labile, cleavable, or reversible linkage is part of a cleavable moiety added between the RNAi agent and the targeting ligand.

In some embodiments, the expression inhibitory oligomeric compound is a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide inhibits the expression of a target mRNA through an RNA interference mechanism. In some embodiments, single-stranded oligonucleotides reduce expression of a target nucleic acid by a mechanism other than RNA interference.

In some embodiments, the gene expression level and/or the target mRNA level in a subject administered a disclosed targeting ligand conjugated to an expression inhibitory oligomeric compound is reduced by at least about 5%, such as by at least about 10%, 15% , 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98 % compared to the subject before administration or the subject not receiving the targeting ligand conjugate. The level of gene expression and/or the level of mRNA in a subject may decrease in a cell, group of cells and/or tissue of the subject. In some embodiments, the protein level of a subject administered a targeting ligand conjugated to an expression inhibitory oligomeric compound is reduced by at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30 %, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to the subject before administration of the conjugate targeting ligand or a subject not receiving the targeting ligand conjugate. A subject's protein level may decrease in a cell, group of cells, tissue, blood, and/or other fluid of the subject. Reductions in gene expression, mRNA or protein levels can be assessed by any methods known in the art. The decrease or decrease in the level of mRNA and/or protein level is collectively referred to as inhibition, reduction or decrease in the expression of the target gene.

Particular expression inhibitory oligomeric compounds that can be used with the disclosed targeting ligands are known in the art. In particular, numerous documents disclose expression inhibitory oligomeric compounds that can be conjugated to the targeting ligands disclosed herein to deliver the composition to the liver. Non-limiting examples include US Patent Application Serial No. 15/281,309 entitled Compositions and Methods for Inhibiting Gene Expression of LPA, which is incorporated herein by reference in its entirety, discloses various double-stranded expression inhibitory oligomeric compounds that target the human apolipoprotein(a) [LPA] gene (to inhibit the expression of the apo(a) protein that is part of the lipoprotein(a) particle, and thus the lipoprotein(a) particle (Lp(a))), which are suitable for use with the targeting ligands disclosed herein. The apo(a) gene [LPA] is expressed in humans and non-human primates, primarily in the liver. Likewise, for example, U.S. Patent Application Serial No. 15/229314 entitled RNAi Therapy for Hepatitis B Virus Infection, which is also incorporated herein by reference, discloses various double-stranded expression inhibitors oligomeric compounds that target the hepatitis B virus, which are suitable for use with the targeting ligands disclosed herein. Hepatitis B virus is a strictly hepatotrophic, double-stranded DNA virus and belongs to the hepadnaviruses belonging to the family Hepadnaviridae. Next as another

- 26045605 example in US patent application serial number 15/229,314 entitled Compositions and Methods for Inhibiting Gene Expression of Factor XII (Compositions and Methods for Inhibiting Gene Expression of Factor XII), which is incorporated herein by reference in its entirety, discloses various double-stranded expression inhibitory oligomeric compounds targeting the Factor XII (or Factor 12, F12) gene, which are suitable for use with the targeting ligands disclosed herein. Factor XII is a serine protease expressed primarily in the liver and present in the blood. Additionally, as another example, US Patent Application Serial No. 14/740307 entitled Compositions and Methods for Inhibiting Gene Expression of Alpha-1 Antitrypsin, which is incorporated herein in its entirety document by reference, disclosed are various double-stranded expression inhibitory oligomeric compounds targeting the alpha-1 antitrypsin (or AAT) gene that are suitable for use with the targeting ligands disclosed herein. AAT is a protease inhibitor belonging to the serpin superfamily, and normal AAT protein is synthesized mainly by liver hepatocytes and secreted into the blood. Further, WO 2016/01123 entitled Organic Compositions to Treat APOC3-Related Diseases, which is incorporated herein by reference in its entirety, discloses various double-stranded expression inhibitory oligomeric compounds targeting human apolipoprotein III (APIII), which are suitable for use with the targeting ligands disclosed herein. Apolipoprotein C-III is a component of lipoproteins that is thought to inhibit the uptake of triglyceride-rich particles by the liver. Additional documents may also be found in the prior art that disclose various therapeutic compounds, including expression inhibitory oligomeric compounds, that may be suitable for use with the targeting ligands disclosed herein. These include, but are not limited to, compositions for which targeting to the liver would be desirable.

Pharmaceutical compositions and formulations

The targeting ligands disclosed herein, when associated with an oligomeric compound, can be used to treat a subject (eg, human or mammal) with a disease or disorder that would benefit from administration of the compound. In some embodiments, the targeting ligands disclosed herein, linked to an expression inhibitory oligomeric compound, can be used to treat a subject (eg, a human) with a disease or disorder in which reduction or inhibition of target mRNA expression would be beneficial. The subject is administered a therapeutically effective amount of any one or more expression inhibitory oligomeric compounds, such as an RNAi agent, that is linked to a targeting ligand disclosed herein. The subject may be a human, a patient, or a human patient. The subject may be an adult, adolescent, child, or infant. The disclosed pharmaceutical compositions comprising a targeting ligand linked to an expression inhibitory oligomeric compound can be used to provide methods for the therapeutic treatment of diseases. Such methods include administering a pharmaceutical composition described herein to a human or animal.

The pharmaceutical compositions and methods disclosed herein can reduce the level of a target mRNA in a cell, group of cells, group of cells, tissue or body of a subject, including: administering to the subject a therapeutically effective amount of an expression inhibitory oligomeric compound described herein that is associated with a targeting ligand, thereby inhibiting expression of the target mRNA in the subject. In some embodiments, the subject has been previously identified as having a pathological upregulation of a target gene in the target cell or tissue.

In some embodiments, the pharmaceutical compositions include at least one expression inhibitory oligomeric compound linked to a targeting ligand. These pharmaceutical compositions are particularly useful in inhibiting the expression of target mRNA in a target cell, group of cells, tissue or organism. The pharmaceutical compositions can be used to treat a subject with a disease or disorder that would benefit from reducing the level of a target mRNA or inhibiting the expression of a target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reducing the level of a target mRNA or inhibiting the expression of a target gene. In one embodiment, the method includes administering a composition comprising a targeting ligand as described herein associated with an expression inhibitory oligomeric compound, such as an RNAi agent, to a subject being treated. In some embodiments, one or more pharmaceutically acceptable excipients (including bases, carriers, diluents, and/or delivery polymers) are added to pharmaceutical compositions comprising a targeting ligand linked to an expression inhibitory oligomeric compound, thereby producing pharmaceutical

- 27 045605 compositions (preparations) suitable for delivery to humans in vivo.

In some embodiments, the disclosed pharmaceutical compositions comprising a targeting ligand linked to an expression inhibitory oligomeric compound are used to treat or regulate clinical manifestations associated with expression of a target mRNA. In some embodiments, a therapeutically or prophylactically effective amount of one or more pharmaceutical compositions is administered to a subject in need of such treatment, prevention, or regulation. In some embodiments, administration of any of the conjugated ligands covalently linked to the oligomeric compound can be used to reduce the number, severity and/or frequency of disease symptoms in a subject.

The described pharmaceutical compositions comprising a targeting ligand linked to an expression inhibitory oligomeric compound can be used to treat at least one symptom in a subject with a disease that would benefit from reducing or inhibiting the expression of the target mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions containing an expression inhibitory oligomeric compound, such as an RNAi agent, linked to a targeting ligand described herein, thereby treating the symptom. In other embodiments, a prophylactically effective amount of one or more expression inhibitory oligomeric compounds is administered to a subject, thereby preventing at least one symptom.

In some embodiments, the level of gene expression and/or the level of target mRNA in a subject administered the expression inhibitory oligomeric compound associated with the targeting is reduced by at least about 5%, such as at least about 10%, 15%, 20% , 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% compared with a subject not receiving the pharmaceutical composition. The level of gene expression in a subject may decrease in a cell, group of cells, and/or tissue of the subject. In some embodiments, the level of mRNA is reduced. In other embodiments, the level of expressed protein is reduced. In some embodiments, the protein level of a subject administered an expression inhibitory oligomeric compound bound to a targeting ligand disclosed herein is reduced by at least about 5%, such as by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% vs. a subject not receiving the pharmaceutical composition. Reductions in expression, mRNA levels, or protein levels can be assessed by any methods known in the art. A decrease or reduction in the level of mRNA and/or protein level is generally referred to herein as inhibition, reduction or decrease in expression of the target gene.

The route of administration is the route by which the expression inhibitory oligomeric compound is brought into contact with the body. In general, methods of administering drugs and nucleic acids to treat a mammal are well known in the art and can be used to administer the compositions described herein. The expression inhibitory oligomeric compound bound to the targeting ligants described herein can be administered by any suitable route in a formulation specifically designed for a particular route. Accordingly, the pharmaceutical compositions described herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intra-articularly or intraperitoneally (intraperitoneally). In some embodiments, pharmaceutical compositions are described and can be administered by inhalation.

Pharmaceutical compositions comprising an expression inhibitory oligomeric compound linked to a targeting ligand described herein can be delivered to a cell, group of cells, tissue or subject using oligonucleotide delivery techniques known in the art. In general, any suitable method for delivering a nucleic acid molecule (in vitro or in vivo) conventional in the art can be adapted for use with the compositions described herein. For example, delivery may be by local administration (eg, direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (eg, intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal administration, inhalation (aerosol), intranasal, rectal, or topical (including buccal and sublingual) administration. In some embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.

Accordingly, in some embodiments, the pharmaceutical compositions described herein may include one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.

As used herein, a pharmaceutical composition or medicament includes pharmacologically

- 28 045605 an effective amount is a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the active pharmaceutical ingredient (API, therapeutic product, e.g., F12 RNAi agent) that are specifically included in a drug delivery system. Excipients do not or should not exhibit a therapeutic effect at the intended dosage. Excipients may serve to a) improve the manufacturability of the drug delivery system during formulation, b) protect, maintain, or increase the stability, bioavailability or usability of the API in a patient, c) facilitate product identification, and/or d) improve one or more of the general safety, effectiveness or delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

Excipients include, but are not limited to: absorption enhancers, antiadhesives, antifoams, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents agents, fillers, flavoring agents, lubricants, humectants, lubricants, oils, polymers, preservatives, brine, salts, solvents, sugars, suspending agents, delayed release matrices, sweeteners, thickeners, tonicity regulators, bases, water repellents and wetting agents.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (for water-soluble substances) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable vehicles for intravenous administration include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ, USA) or phosphate buffered saline (PBS). They must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyhydric alcohol (eg, glycerin, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. In many cases, it is preferable that the composition includes isotonic agents, for example, sugars, polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injection solutions can be prepared by incorporating the active compound into the required amount of a suitable diluent with one or a combination of the ingredients listed above, if necessary, followed by sterilization by filtration. Typically, dispersions are prepared by introducing the active compound into a sterile vehicle that contains the basic dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the production method includes vacuum drying and freeze drying, which allows the powder of the active ingredient in combination with any additional desired ingredient to be obtained from a solution thereof previously subjected to sterile filtration.

Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of the drug, which may be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Lysosomal formulations or systems including a biodegradable polymer can also be used to prepare a drug for both intra-articular and ocular administration.

Forms suitable for topical administration, including eye treatments, include liquid or semi-liquid preparations such as liniments, lotions, gels, application forms, oil-in-water and water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions, such as drops. Formulations for topical application to the surface of the skin can be prepared by dispersing the drug in a dermatologically acceptable carrier such as lotion, cream, ointment or soap. Suitable carriers are capable of forming a film or layer on the skin that allows localized application and reduces movement. For topical administration to internal tissue surfaces, the agent may be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to the tissue surface. For example, the use of hydroxypropylcellulose or fibrinogen/thrombin solutions may be useful. Alternatively, fabric coatings can be used in coatings, such as pectin-containing formulations.

For inhalation treatments, powder inhalation (self-propelling or spray formulations) can be used and can be dosed using an aerosol can,

- 29 045605 nebulizer or atomizer. Such formulations may be presented in fine powder form for pulmonary administration from a powder inhalation device or in self-propelling forms for powder administration. In the case of self-propelling solutions or spray forms, In the case of self-propelling solutions or spray forms, the desired effect can be achieved either by selecting a valve with the desired spray characteristics (i.e., capable of dispensing a spray of the desired particle size) or by introducing the active ingredient as suspended powder into controlled particle sizes. For administration by inhalation, the compounds may also be administered in the form of an aerosol spray from a container or dispenser that contains a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.

Systemic administration may also be via mucosal or transdermal routes. For mucosal or transdermal administration, penetrants suitable for the barrier to be crossed are used in the form. Such penetrants are well known in the art and include, for example, mucous membrane penetrants, detergents and bile salts. Administration through mucous membranes can be accomplished using nasal sprays or suppositories. For transdermal administration, the active compounds are typically formulated into ointments, ointments, gels or creams, as is widely known in the art.

The active compounds can be combined with carriers that will protect the compound from rapid elimination from the body, such as sustained release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such compositions will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. They can be prepared in accordance with methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

Compositions for oral or parenteral use may be in dosage form for ease of administration and uniformity of dosage. Dosage form refers to physically discrete units that can be used as dosage units for a subject being treated; each unit contains a predetermined amount of active compound calculated to provide the desired therapeutic effect in combination with the required pharmaceutical carrier. The properties of the dosage forms of the present invention are dictated by and directly depend on the unique characteristics of the active compound and the therapeutic effect intended to be obtained, and the inherent limitations associated with combining such an active compound with other components for the treatment of individuals. In addition, administration may be accomplished by intermittent bolus injections, or may be more continuous using intravenous, intramuscular, or intraperitoneal administration of their external reservoir (eg, intravenous bag).

In combination with the methods of the present disclosure, pharmacogenomics (ie, the study of the relationship between an individual's genotype and the individual's response to a foreign compound or drug) can be considered. Differences in the metabolism of therapeutic agents can lead to severe toxic effects or treatment failure by altering the relationship between dose and plasma concentration of the pharmacologically active drug. Accordingly, the attending physician or clinician may consider using information obtained from appropriate pharmacogenomic studies to determine whether to administer a drug and to tailor the dosage and/or therapeutic regimen of the drug.

Pharmaceutical composition other additional components typically present in pharmaceutical compositions. Such additional components include, but are not limited to: antipruritics, astringents, local anesthetics or anti-inflammatory agents (eg, antihistamine, diphenhydramine, etc.). It is also contemplated that cells, tissues, or isolated organs that express or contain RNAi agents as defined herein can be used as pharmaceutical compositions. As used herein, a pharmacologically effective amount, a therapeutically effective amount, or simply an effective amount refers to that amount of an RNAi agent that produces the intended pharmacological, therapeutic, or prophylactic result.

Typically, an effective amount of the active compound will range from about 0.1 to about 100 mg/kg body weight/day, for example from about 1.0 to about 50 mg/kg body weight/day. In some embodiments, the effective amount of the active compound will range from about 0.25 to about 5 mg/kg body weight per dose. In some embodiments, the effective amount of the active ingredient will range from about 0.5 to about 3 mg/kg body weight per dose. The amount administered will also likely depend on variables such as the general health of the patient, the relative biological effectiveness of the compound delivered, the type of drug formulation, the presence and type of excipients in the formulation, and the route of administration. In addition, it should be understood that the initial dosage administered may be increased above an upper limit to rapidly achieve the target blood level or tissue level, or the initial dosage may be less than the optimal value.

To treat a disease or prepare a drug or composition for treating a disease, the pharmaceutical compositions described herein comprising an expression inhibitory oligomeric compound, such as an RNAi agent, linked to a targeting ligand, can be combined with an excipient or with a second therapeutic agent or treatment, including, but not limited to, a second or other expression inhibitory oligomeric compound, small molecule drug, antibody, antibody fragment, and/or vaccine.

The disclosed targeting ligands coupled to expression inhibitory oligomeric compounds and added to pharmaceutically acceptable excipients or adjuvants can be packaged in kits, containers, packages or dosing devices. The pharmaceutical compositions described herein may be packaged in pre-filled syringes or ampoules.

Below, the embodiments proposed above are described by way of non-limiting examples.

Examples

The following examples are not limiting and are intended to illustrate some of the embodiments disclosed herein.

Some of the abbreviations used in describing the experimental details of the synthesis of the example compounds described below are defined below: h = hour(hours); min = minute(s); mole = mol(moles); mmol = millimole (millimoles); M = molar; µM= micromolar; g = gram(grams); micro = microgram (micrograms); rt or RT = room temperature; ct l = liter (litres); ml = milliliter(milliliters); wt. = mass; Et2O = diethyl ether; THF = tetrahydrofuran; DMSO=dimethyl sulfoxide; EtOAc = ethyl acetate; Et ₃ N or TEA = triethylamine; i-Pr2NEt, or DIPEA, or DIEA = diisopropylethylamine; CH2C12 or DCM = methylene chloride; CHCl ₃ = chloroform; CDCl ₃ = deuterated chloroform chloroform; CCl ₄ = carbon tetrachloride; MeOH = methanol; EtOH = ethanol; DMF = dimethylformamide; BOC = tert-butoxycarbopyl; CBZ = benzyloxycarbonyl; TBS = t-butyldimethylsilyl; TBSCl = tbutyldimethylsilyl chloride; TFUK. = trifluoroacetic acid; DMAP = 4-dimethylaminopyridine; NaN ₃ = sodium azide; Na ₂ SO ₄ = sodium sulfate; NaHCO ₃ = sodium bicarbonate; NaOH = sodium hydroxide; MgSO ₄ = magnesium sulfate; K2CO3 = potassium carbonate; KOH = potassium hydroxide; NH4OH = ammonium hydroxide; NH4CI = ammonium chloride; SiO2 = silicon oxide Pd-C = palladium on carbon; HCl = hydrogen chloride or hydrochloric acid; NMM = N-methylmorpholine; H2 = hydrogen gas; KF = potassium fluoride; EDC-HCl = N-(3Dimethylaminepropyl)-N'-ethylcarbodiimide hydrochloride; MTBE = methyl tert-butyl ether; MeOH = methanol; Ar = argon; SiO2 = silicon oxide RT = retention time.

Additionally, examples of expression inhibitory oligomeric compounds suitable for use with the targeting ligands disclosed herein are provided in various tables in the Examples section below. To designate modified nucleotides for the sequences shown in the tables below, the following designations are used:

N = 2'-OH (unmodified) ribonucleotide (capital letter without f or d) n = 2'-OMe modified nucleotide

Nf = 2'-fluoro-modified nucleotide dN = 2'-deoxynucleotides

Nuna = 2',3'-seconucleotide mimetics (uncapped nucleoside analogues)

NIna = closed nucleotide

Nf _ANA = 2'-D-arabinonucleotide

NM = 2'-methoxyethyl nucleotide

X or Ab = ribose without nucleoside base

R = ribitol (invdN) = inverted deoxyribonucleotide (3'-3'-linked nucleotide) (invAb) = inverted nucleotide without nucleoside base (invX) = inverted nucleotide without nucleoside base (invn) = inverted 2'-OMe nucleotide s = phosphorothioate -bound nucleotide vpdN = vinylphosphonate deoxyribonucleotide (3'OMen) = 3'-OMe-nucleotide (5Me-Nf) = 5'-Me, 2'-fluoronucleotide cPrp = cyclopropylphosphonate

The compounds disclosed herein can be manufactured using chemical

- 31 045605 techniques known to specialists in this field.

Example 1. Synthesis of a targeting ligand representing a forforamidite compound of structure 101b.

1) Preparation of tri-tert-butyl-N-[N-(benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamate (3)

Compound 1 (10.00 g, 29.64 mmol) was added to a nitrogen-purged 250 mL three-neck round bottom flask equipped with a thermocouple, magnetic stirrer, nitrogen inlet, and powder funnel, followed by THF (100 mL). The resulting solution was stirred and then N-methylmorpholine (7.82 ml, 71.15 mmol) was added.

The powder funnel was replaced with a rubber septum and the mixture was cooled using an ice bath to 0°C. Isobutyl chloroformate (iBuCOCl, 3.85 mL, 29.64 mmol, 1.0 eq.) was added dropwise to the reaction mixture over 10 minutes, maintaining the temperature in the vessel below 4.0°C. After addition, the mixture was stirred for another 40 minutes and the partition was replaced with a powder funnel. Compound 2 (8.767 g, 29.64 mmol, 1.0 eq.) was added to the reaction mixture in portions over 15 min, maintaining the temperature in the vessel below 4.0°C. After adding Compound 2, the ice bath and powder funnel were removed and the reaction mixture was allowed to warm to ambient temperature for the remaining steps. The clear, colorless solution was kept for 25 minutes after adding compound 2.

A sample of the reaction mixture (98 μL diluted in 5.0 mL of acetonitrile in a 5 mL volumetric flask) was taken 40 min after the start of the addition of compound 2 and the percent conversion was examined by RP-HPLC. 23% of compound 1 was found to remain, so 60 min after the start of the reaction, additional iBuCOCl (1.16 ml, 30 mol%) and 2 (2.63 g, 30 mol%) were added sequentially. The solution was kept for an additional 60 minutes until HPLC indicated that the sample showed more than 99% conversion. The total reaction time was 2.5 hours from the start of the first addition of compound 2.

The reaction solution was poured into a stirred solution of 0.5 M HCl (aq), cooled in an ice bath to 3°C, and stirred for approximately 5 min. The quenched reaction mixture was transferred to a 500 mL separatory funnel and ethyl acetate (100 mL) was added. The layers were separated and the organic phase was washed with brine (100 ml), dried with MgSO ₄ , filtered into a 500 ml round bottom flask and concentrated in vacuo to give a thick, colorless oil. This oil was dissolved in MTBE (100 ml) and concentrated in vacuo to again give a thick, colorless oil.

Hexanes (100 ml) were added to the stirred oil. A white cloud appeared in the solution, which disappeared after stirring. The seed crystals were added and the mixture was left to stir for 40 min; During this time, crystals slowly formed.

Within 20 minutes the suspension became thick enough to make stirring difficult and additional (50 ml) was added. After 40 min, the suspension was filtered using a large-pore funnel, washed three times with hexanes (~10 ml in each case) and air-dried in the funnel for 1 h, resulting in compound 2 as a fine white powder (15.64 g, 91%) . ¹ H-NMR of compound 3 is shown in FIG. 1. On a 75g scale, the yield was 917% with a purity of 99%.

2) Preparation of N-[N-(benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamic acid (4)

Compound 3 (72.57 g, 125.4 mmol) and formic acid (analytically pure, >95%, 1.45 L, 20 vol equiv) were added to a 3000 mL three-neck round bottom flask equipped with an overhead stirrer, powder funnel, thermocouple, and heating mantle. . The funnel for the powder was replaced with a stopper/N ₂ and the resulting solution was heated to 45 °C and stirred for 1 hour, monitoring the progress of the reaction by RP-HPLC. The reaction was considered complete when less than 2.0% of the area of mono-t-butyl ethers remained.

A sample of the reaction mixture (50 µl, diluted in 950 µl H ₂ O) was taken 60 minutes after the addition of formic acid, and the sample was examined by RP HPLC to determine the percentage of mono-t-butyl ether remaining. Analysis showed that 1.8% mono-t-Bu-esters remained; Accordingly, heating was stopped for 90 min.

The reaction mixture was diluted with toluene and acetonitrile (ACN, 1500 ml in each case) and the mixture was concentrated in vacuo. Formic acid was removed by azeotroping with 1:1 ACN:toluene (−600 mL) twice with ACN (−500 mL in each case). This material was dried under high vacuum overnight to provide 4 as a white foamy solid (54.3 g, quantitative yield). ¹ H-NMR of compound 4 (L/N 1321-063B) is shown in FIG. 2.

3) Preparation of N-[N-(benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamic acid, tpu-[NAGPEG ₂ ]amide (6)

NAG-amine p-tosylate salt (5, 59.19 g, 97.6 mmol, 4.13 equiv.) and 2-bis-Glu-triacide (4, 10.01 g, 23.6 mmol, 1.0 equiv.) were added to a 1 L round-bottom flask. The mixture was dissolved in acetonitrile (500 ml) and concentrated under vacuum to azeotropically remove water. The residue was dissolved in fresh acetonitrile (400 ml) and transferred to a nitrogen-purged three-neck round bottom flask equipped with a stirrer and equipped with a thermocouple. Water content was measured by the Karl Fischer method (257 ppm).

TBTU (28.20 g, 87.8 mmol, 3.7 eq.) was added to the stirred solution through a powder funnel under nitrogen atmosphere. The remaining TBTU on the funnel was washed into the reaction mixture using additional acetonitrile (100 ml). DIPEA (34.0 mL, 25.2 g, 8.0 eq.) was added dropwise via syringe over 20 min, maintaining the reaction temperature below 25°C. The mixture was stirred for 2 hours from the start of the addition of DIPEA, monitoring by HPLC. At 78 min the analysis showed complete absorption of the starting material.

After 2 h, the solvent was removed under vacuum. The resulting thick oil was dissolved in dichloromethane (1000 ml) and washed with 1.0 N. HCl (aq) (3x500 ml) and saturated NaHCO ₃ (aq) (3x500 ml). The organic layer was dried with Na ₂ SO ₄ , filtered and concentrated in vacuo to give a whitish waxy solid (33.5 g).

Flash chromatography was carried out on an ISCO CombiFlash automated purification system using chloroform and methanol as eluents. All fractions suspected of containing product based on the UV chromatogram (220 nm) were analyzed by HPLC, and all fractions containing at least 97.0% AUC of product were combined and concentrated to yield 18.75 g (97.0% purity) of compound 6. The fractions with impurities were combined, resulting in another 12.2 g (78.8% purity) of compound 6. The total yield of 6 was 70.9%. ¹ H-NMR of compound 6 is shown in FIG. 3.

4) Preparation of tpu-NAG-6uc-G1u-NH ₂ tosylate (7).

Compound 6 (5.737 g, 3.46 mmol) in MeOH (155 ml) with p-TsOH-H ₂ O (0.657 g, 3.46 mmol) was hydrogenated in the presence of Pd/C 10% (688 mg) for 6 h. TLC (CHCl ₃ ;MeOH = 8.5:1.5) confirmed that by this time the reaction was complete. The reaction flask was filled with Ar, EtOH (200 ml) was added and the solution was filtered through celite. The product was concentrated and dried under vacuum. 4.81 g of the desired tosylate salt 7 were obtained. 1H-NMR of compound 7 is shown in FIG. 4.

5) Preparation of tpu-NAG-6uc-G1u-NH-PEG ₆ -OH (9):

- 33 045605

Procedure A (if the purity of mpu-NAG amine salt 7 is less than 96%): NAG-amine salt 7 (purity ~90%, 18.50 g, 10.90 mmol) and HO-PEG6-CO2TFP - 8 ester (6.57 g, 13.08 mmol) were dissolved in dichloromethane (185 ml) and cooled to 0°C. Triethylamine (6.10 mL, 43.59 mmol) was added to this solution. The solution was allowed to warm to room temperature and stirred for 18 hours while being monitored by HPLC. The reaction was quenched with saturated aqueous NaHCO ₃ and brine (1:1, 140 ml), stirred for 30 min at room temperature, and the layers were separated. The organic layer was washed with saturated aqueous NaHCO ₃ (3x140 ml) and brine (1:1) and dried with Na ₂ SO ₄ . The drying agent was filtered and the solution was concentrated and purified by flash chromatography to give compound 9 (13.56 g, 67%) as a white solid. ¹ H-NMR of compound 9 is shown in FIG. 5.

Flash chromatography was carried out on an ISCO CombiFlash automated purification system using dichloromethane and methanol as eluents. The pure fractions were combined and concentrated to yield 13.56 g of compound 9 (99% purity). Fractions with impurities were combined, resulting in 4.9 g of compound 9 (purity ~95%).

Procedure B (if the purity of the mpu-NAG-amine 7 salt is greater than 96%):

Product 7 (1.94 g, 1.272 mmol) in DCM (40 ml) was stirred under argon with HOPEG6-CO2TFP ether 8 (767 mg, 1.526 mmol) and DIPEA (443 μl, 2.544 mmol) for 16 h. The reaction mixture was concentrated under vacuum, dissolved in CHCl ₃ and added dropwise to stirred Et ₂ O (90 ml). The precipitate was separated, rinsed with Et ₂ O (3x35 ml) and dried under vacuum. Yield 2.275 g (96%).

6) Preparation of tpu-NAG-6uc-G1u-NH-PEG ₆ phosphoramidite (10):

Compound 9 (6.62 g, 3.56 mmol) and 4,5-dicyanoimidazole (0.11 g, 0.89 mmol) were dissolved in anhydrous dichloromethane (230 ml) and placed under a nitrogen atmosphere. To this mixture was added dropwise a solution of 2cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidide (Phos reagent, 1.46 mL, 4.62 mmol) in anhydrous dichloromethane (5 mL) over 5 min. The reaction mixture was stirred at room temperature for 3 h without HPLC monitoring (<1% starting material remained).

The reaction mixture was washed with saturated aqueous NaHCO ₃ (2x150 ml), 3% DMF in H2O (v/v, 2x150 ml), H2O (3x150 ml) and brine (1x150 ml), the organic layer was dried using Na ₂ SO ₄ . The drying agent was filtered and the solution was concentrated in vacuo to obtain the crude product. The crude product was suspended in 5% toluene-hexane (50 ml) and stirred for 5 minutes, after which the solvent was discarded. The process was repeated with 5% toluene-hexane (1x50 ml) and hexane (2x50 ml). The solids were dried under vacuum to obtain 6.69 g of 10 in the form of a white solid (91%) (compound 10). ¹ H-NMR of compound 10 (Structure 101d herein) is shown in FIG. 6.

Example 2. Synthesis of a targeting ligand representing a forforamidite compound of structure 103d.

1) Preparation of tpu-NAG-6uc-G1u-NH-PEG4-OH (12):

- 34 045605

Product 7 (2.44 g, 1.44 mmol) from Example 1 above was dissolved in DCM (30 ml) and placed under argon. HO-PEG4-CO2TFP ester 11 (717 mg, 1.73 mmol) and DIPEA (502 μL, 2.88 mmol) were added to the solution. The resulting mixture was stirred for 16 hours. The reaction mixture was concentrated in vacuo and redissolved in CHCl ₃ . The solution was then added dropwise to stirred Et ₂ O (90 ml). The precipitate was separated, rinsed with Et ₂ O and dried under vacuum, resulting in 2.60 g (102%) of product 12, which was used without further purification.

2) Preparation of tpu-NAG-6uc-G1u-NH-PEG ₄ phosphoramidite (13):

Product 12 (1.80 g, 1.01 mmol) was evaporated twice with pyridine and then dissolved in anhydrous dichloromethane (25 ml) and placed under argon. Diisopropylammonium tetrazolide (87 mg, 0.51 mmol) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidide (458 mg, 1.52 mmol) were added to the solution. The reaction mixture was stirred at room temperature for 5 hours, monitoring the progress of the reaction by TLC (CHCl ₃ : MeOH: Et ₃ N 95:5:2). After uptake of the starting material, the reaction mixture was diluted with dichloromethane (250 ml) and washed with saturated aqueous NaHCO ₃ (100 ml) and saturated aqueous brine (100 ml). The organic layer was dried with sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography (DCM:MeOH:Et ₃ N 97:3:2) to give 1.04 g (53%) of compound 13. ¹ H-NMR of compound 13 (Structure 103d herein) is shown in FIG. 7.

Example 3. Synthesis of a targeting ligand representing a forforamidite compound of structure 102d

1) Preparation of tpu-NAG-6uc-G1u-NH-PEG ₈ -OH (15):

Product 7 (3.09 g, 1.82 mmol) from Example 1 above was dissolved in DCM (30 ml) and placed under argon. HO-PEG ₈ -CO ₂ TFP 14 ester (1.29 g, 2.18 mmol) and DIPEA (634 μl, 3.64 mmol) were added to the solution. The resulting mixture was stirred for 16 hours. The reaction mixture was concentrated in vacuo and redissolved with CHCl ₃ . The solution was then added dropwise to stirred Et2O (180 ml). The precipitate was separated, rinsed with Et2O and dried under vacuum, resulting in 3.54 g (99%) of product 15, which was used without further purification.

2) Preparation of tpu-NAG-6uc-G1u-NH-PEG ₈ phosphoramidite (16):

Product 15 (1.79 g, 0.92 mmol) was evaporated twice with pyridine and then dissolved in anhydrous dichloromethane (25 ml) and placed under argon. Diisopropylammonium tetrazolide (79 mg, 0.46 mmol) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidide (416 mg, 1.38 mmol) were added to the solution. The reaction mixture was stirred at room temperature for 3 hours, monitoring the progress of the reaction by TLC (CHCl ₃ : MeOH: Et ₃ N 95:5:2). After uptake of starting material, the reaction mixture was concentrated in vacuo and redissolved in DCM. The solution was then added dropwise to stirred Et ₂ O (90 ml). The precipitate was separated, rinsed with Et ₂ O and dried. The crude material was purified by column chromatography (CHCl ₃ : MeOH: Et ₃ N 97:3:2), resulting in

- 35 045605

950 mg (48%) of compound 16. 1H-NMR of compound 16 (Structure 102d herein) is shown in FIG. 8.

Example 4. Synthesis of an oligonucleotide composition.

A. Synthesis, RNAi agents were synthesized according to the solid phase phosphoramidite technology used in the synthesis of oligonucleotides. Depending on the scale, either MerMade96E® (Bioautomation) or MerMadel2® (Bioautomation) was used. The synthesis was carried out on a solid support made of controlled pore glass (CPG, 500A or 600A, obtained from Prime Synthesis, Aston, PA, USA). All RNA and 2′-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). In particular, the following 2'-Omethylphosphoramidites were used: (5'-O-dimethoxytrityl-K ⁶ -(benzoyl)-2'-O-methyladenosine-3'-O-(2-cyanoethyl-K,Kdiisopropylamino)phosphoramidite, 5' -O-dimethoxytrityl-K ⁴ -(acetyl)-2'-O-methylcytidine-3'-O-(2-cyanoethyl-K,K-diisopropylamino)phosphoramidite, (5'-O-dimethoxytrityl-K ² -(isobutyryl )-2'-O-methylguanosine-3'-O-(2-cyanoethyl-K,K-diisopropylamino)phosphoramidite and 5'-O-dimethoxytrityl-2'-O-methyluridine-3'-O-(2-cyanoethyl -K,K-diisopropylamino)phosphoramidite. 2'-deoxy-2'-fluorophosphoramidites carried the same protecting groups as 2'-O-methyl-RNA amidites. Targeting ligands containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), and the other amidites were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3A) were added. in acetonitrile) was used as the activating solution.The binding times were 10 min (RNA), 15 min (targeting ligand), 90 sec (2'OMe) and 60 sec (2'F). To introduce phosphorothioate bonds, a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

B. Cleavage and deprotection of the oligomer bound to the support.

After completion of the solid-phase synthesis, the dried solid support was treated with a 1:1 v/v solution of 40 wt.% methylamine in water and 28% ammonium hydroxide solution (Aldrich) for two hours at 30°C. The solution was evaporated and the solid residue was reduced in water (see below).

C. Cleaning.

Crude oligomers were purified by anion exchange HPLC using a TKSgel SuperQ-5PW 13u column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, and contained 20% acetonitrile, and buffer B was the same as buffer A but with the addition of 1.5 M sodium chloride. Residual UV was recorded at 260 nm. The appropriate fractions were pooled and then subjected to exclusion HPLC using a GE Healthcare XK 16/40 column packed with Sephadex G-25 running buffer of 100 mM ammonium bicarbonate, pH 6.7, and 20% acetonitrile.

D. Annealing.

The complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form RNAi agents. This solution was placed in a thermomixer at 70°C, heated to 95°C, kept at 95°C for 5 minutes and slowly cooled to room temperature. Some RNA agents were lyophilized and stored at -15 to -25°C. The concentration of duplexes was determined by measuring the absorbance of the solution using UV-Vis. spectrometer in 0.2x FBI. The absorbance of the solution at 260 nm was then multiplied by the conversion factor and the dilution factor, thereby determining the duplex concentration. Unless otherwise stated, the conversion factor was 0.037 mg/(ml-cm) in all cases. For some experiments, the conversion coefficient was calculated from the experimentally determined extinction coefficient.

Example 5. Properties of phosphoramidite-containing compounds including targeting ligands with PEG linkers of varying lengths

The following phosphoramidite compounds with targeting ligands were synthesized according to the methods described above in Examples 1-4:

- 36 045605

Each of the phosphoramidite compounds Structures 101d, 102d and 103 d was introduced in an amount of 16 equiv. to conjugate at the 5' end of the single-stranded oligonucleotide AM03704-SS, which is a sense strand that can be used in the synthesis of a double-stranded RNA interference (RNAi) agent targeting F12. AM03704 has the nucleotide sequence shown in the table below:

Table 1. Sequence of the semantic chain from example 5

The compositions were solubilized in dichloromethane (DCM) and dried over sieves. For the phosphoramidite compound of structure 103d (i.e. containing the PEG-4 linker), gelation problems were observed at both 0.05M and 0.25M concentrations. As shown in FIG. 9, under these conditions, only a very small amount of the targeting ligand of Structure 103 d was able to conjugate to the 5' end of the AM03704-SS oligonucleotide.

Conjugation to the targeting ligand oligonucleotide of both Structure 101d and Structure 102d was observed. In fig. Figure 9 shows the HPLC chromatogram for AM03704 conjugated to Structure 101d. It was determined that in the case of the targeting ligand Structure 101, approximately 78% of the targeting ligand-conjugated oligonucleotide (FLP = full-length product) was generated. In fig. 10 shows the HPLC chromatogram for AM03704 conjugated to Structure 102d. Approximately 40% of the oligonucleotide was conjugated to the targeting ligand and approximately 60% of the oligonucleotide remained unconjugated.

In a surprising and unexpected way, with the introduction of 16 eq. Structure 101d was substantially superior to both Structure 102d and Structure 103d for conjugation to an oligonucleotide at the 5' end of the sequence. In addition, both Structure 101d and 102d exhibited higher solubility compared to Structure 103d. As noted above, Structure 103d was poorly soluble using standard concentrations and solvents typical for oligonucleotide synthesis. The production of targeting ligands coupled to expression inhibitory oligomeric compounds containing the targeting ligand of Structure 103 (using the phosphoramidite compound of Structure 103d) required the addition of more aggressive polar solvents.

Example 6: Comparison of 3' and 5' Sense Strand Attachment Sites for GalNAc Targeting Ligands Using F12 Expression Inhibiting Oligomeric Compounds in Wild-Type Mice

To assess differences in the attachment site of GalNAc ligands between the 3' and 5' ends of the sense

- 37 045605 chains were prepared with expression inhibitory oligomeric compounds (double strand RNAi agents) targeting F12 (referred to herein as F12 RNAi agents) with the sequences presented in Table 1 below. 2:

Table 2. F12 expression inhibitory oligomeric compounds (RNAi agent duplexes) from Example 6

Duplex ID: AD02803 5' 3' SEQ ID NO: Sequence of the semantic chain: (AM03628-SS) uAuAugscsccaagaAfaGfugaaagacca(NAG15) 2 Antisense strand sequence: (AM03157-AS) usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 3 Duplex ID: AD02807 5’ -» 3’ SEQ ID NO: Sequence of the semantic chain: (AM03632-SS) (NAG18)uauaugscsccaagaAfaGfugaaagacc(invdA) 4 Antisense strand sequence: (AM03157-AS) usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 5

In table 2 above the following notations are used:

(NAG18) has a chemical structure that is represented herein by Structure 2.

Each F12 RNAi agent strand was synthesized using phosphoramidite technology on solid phase oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 4 herein.

F12 RNAi agents bound to the appropriate GalNAc ligand (ie (NAG15) or (NAG18)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

F12 RNAi agents bound to appropriate GalNAc ligands were delivered via s.c. injection. On day 1, a solution containing either saline or a dose of 3 mg/kg (milligrams per kilogram - mpk) of one of two agents for RNAi F12 ( AD02803 or AD02807) in buffered saline. Three (3) wild-type mice per treatment group were used. As shown above, AD02803 includes (NAG15) bound to the 3' end of the sense strand, while AD 2807 includes (NAG18) bound to the 5' end of the sense strand.

Serum samples from treated mice were collected on days 8, 15, 22, and 29 to monitor knockdown. Knockdown was measured by quantifying circulating F12 protein levels

- 38 045605 mouse (mF12) in serum using a proprietary mF12 assay developed based on alphaLISA® (Perkin Elmer). Expression corresponding to a specific date of blood collection was normalized to the mean value corresponding to the same date for the saline control group.

In fig. 12 presents the results of this study. At trough levels (day 22), AD02803 showed an approximately 70% reduction in circulating F12 levels, while AD02807 showed a greater than 80% reduction. These data also indicate a difference in the duration of the knockdown effect, as at day 29, AD02803-treated mice showed a more rapid return to basal levels compared to AD2807-treated mice.

These data confirm that GalNAc ligand binding at the 5' end of the sense strand is superior to binding at the 3' end of the sense strand.

Example 7'. Additional comparison of 3' and 5' sense strand attachment sites for GalNAc targeting ligands using F12 expression inhibitory oligomeric compounds in wild-type mice

To further evaluate the attachment site of GalNAc ligands at the 3' and 5' ends of the sense strand of double-stranded expression inhibitory oligomeric compounds (double-stranded RNAi agents), compositions targeting the F12 gene were prepared with the sequences presented in Table 1 below. 3.

Table 3. F12 expression inhibitory oligomeric compounds (RNAi agent duplexes) from Example 7

Duplex ID: AD02815 5' ^3' SEQ ID NO: Sequence of the semantic chain: (AM03640-SS) (NAG20)uauaugscsccaagaAfaGfugaaagacc(invdA) 6 Antisense strand sequence: (AM03157-AS) usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 7 Duplex ID: AD02816 5' 3' SEQ ID NO: Sequence of the semantic chain: (AM03641-SS) uAuAugscsccaagaAfaGfugaaagacca(NAG20) 8 Antisense strand sequence: (AM03157-AS) usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 9

In table 3 above the following notation is used: (NAG20)=

(NAG20) has a chemical structure that is represented herein by Structure 4.

Each strand of F12 RNAi agents was synthesized using solid-phase phosphoramidite technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 4 herein.

F12 RNAi agents bound to the appropriate GalNAc ligand (ie (NAG20)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

F12 RNAi agents bound to the appropriate GalNAc ligand were delivered via

- 39 045605 subcutaneous injection. On day 1, 200 μl/20 g of mouse body weight of a solution containing either saline or a dose of 3 mg/kg (mpk) of one of two RNAi agents (AD02815 or AD02816) was injected subcutaneously into the fold of skin on the back between the shoulder blades. in buffered saline solution. Three (3) wild-type mice were used per treatment group. As shown in the table above. 3, AD02815 includes (NAG20) bound to the 5' end of the sense strand, while AD02816 includes (NAG20) bound to the 3' end of the sense strand.

Serum samples from treated mice were collected on days 8, 15, 22, and 29 to monitor knockdown. Knockdown was measured by quantifying circulating levels of mouse F12 protein (mF12) in serum using an in-house mF12 assay developed based on alphaLISA® (Perkin Elmer). Expression corresponding to a specific date of blood collection was normalized to the mean value corresponding to the same date for the saline control group.

In fig. 13 shows the results of this experiment. At trough levels (day 22), AD02816 showed an approximately 60% reduction in circulating F12 protein levels, whereas AD02815 showed a 79% reduction. These data also indicate differences in the duration of the knockdown effect. At day 29, AD02816-treated mice showed 40% knockdown, while AD02815-treated mice showed 71% knockdown relative to saline levels. These data confirm the binding of the GalNAc ligand at the 5' end of the sense strand.

Example 8. LP(a) Expression Inhibiting Oligomeric Compounds (Double Stranded RNAi Agents) Bound to Structure 101 Targeting Ligands in Lp(a) Transgenic (Tg) Mice

Oligomeric compounds inhibiting the expression of LP(a) (double-stranded Lp(a) RNAi agents) were prepared with the sequences presented in Table 1 below. 5.

Table 4. LP(a) expression inhibitory oligomeric compounds (RNAi agent duplexes) from Example 8

Duplex ID: AD03547 5' 3' SEQ ID NO: Sequence of the sense chain: (AM04498-SS) (NAG29)uauauaasuuaucgaGfGfcucauucucsa(invAb) 10 Antisense strand sequence: (AM04507-AS) usGfsasGfaAfuGfaGfccuCfgAfuAfausuAUAUA eleven Duplex ID: AD03549 5' ^3' SEQ ID NO: Sequence of the sense chain: (AM04502-SS) (NAG25)uauauaasuuaucgaGfGfcucauucucsa(invAb) 12 Antisense strand sequence: (AM04507-AS) usGfsasGfaAfuGfaGfccuCfgAfuAfausuAUAUA 13

In table 4 above the following notations are used:

- 40 045605

(NAG25) has a chemical structure that is represented herein by Structure 101.

Each strand of Lp(a) RNAi agents was synthesized according to solid-phase phosphoramidite technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 4 herein.

Lp(a) transgenic (Tg) mice were used to evaluate the effectiveness of double-stranded RNAi agents with conjugated N-acetylgalactosamine ligands in vivo (Frazer KA et al 1995, Nature Genetics 9:424-431). These mice (hereinafter referred to as Lp(a) Tg mice) express human apo(a) with a YAC containing the complete LPA gene (encoding the apo(a) protein) with additional sequences in both the 5' and in the 3' direction, as well as human apoB100, thereby producing humanized Lp(a) particles (Callow MJ et al 1994, PNAS 91:2130-2134).

Lp(a) RNAi agents bound to appropriate GalNAc ligands (ie (NAG25) or (NAG29)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

Lp(a) RNAi agents bound to the corresponding GalNAc ligands (i.e. (NAG25) or (NAG29)) at the 5′ end of the sense strand were delivered by SC injection. On day 1, a solution containing either saline or a dose of 1 mg/kg (mpk) of the appropriate agent for Lp(a) RNAi (AD03547 or AD03549) in buffered saline. Four (4) Lp(a) Tg mice were used per treatment group.

Serum samples from treated mice were collected on days -1 (pre-dosing), 5, 11, 16, 22, 29 and 36. Knockdown was determined by calculating serum levels of circulating Lp(a) particles. Lp(a) particle levels were measured on a Cobas® Integra 400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by the pre-dosing expression level in that animal (in this case, the -1 day level) to determine the expression ratio normalized to -1 day.

Expression at a specific time point was then normalized to the saline control group by dividing the -1 day normalized coefficient for an individual animal by the average of the -1 day normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. The experimental error is presented as standard deviation.

The results are shown in Fig. 14. For AD03549 (NAG25), 71% knockdown was observed at minimum values (day 16); for AD03547 (NAG29), 81% knockdown was observed at trough values (day 11). Both triggers showed similar recovery curves from trough values, with less than 26% knockdown at day 36. These data confirm that the GalNAc ligands shown were distinguished by both comparable initial knockdown activity and comparable

- 41 045605 duration of knockdown in Lp(a) Tg mice with a single dose of 1 mg/kg.

Example 9. Knockdown of Lp(a) in Lp(a) transgenic (Tg) mice after administration of LP(a) expression-inhibiting oligomeric compounds (double-stranded RNAi agents) bound to a targeting ligand Structures 101

Oligomeric compounds inhibiting the expression of LP(a) (double-stranded Lp(a) RNAi agents) were prepared with the sequences presented in Table 1 below. 5:

Table 5. LP(a) expression inhibitory oligomeric compounds (RNAi agent duplexes) from Example 9

Duplex ID: AD03272 5' 3' SEQ ID NO: Sequence of the semantic chain: (AM04138-SS) (NAG25)uauausasguuaucgAfGfGfcucauucuc(invdA) 14 Antisense strand sequence: (AM02860-AS) usGfsaGfaAfuGfaGfccuCfgAfuAfaCfucsusuAu 15

In table 5 (NAG25) is represented by the same structure as shown in Example 8 above and has a chemical structure that is represented herein by Structure 101.

Each strand of Lp(a) RNAi agents was synthesized according to solid-phase phosphoramidite technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 4 herein.

Lp(a) Tg mice were used to evaluate the effectiveness of double-stranded RNAi agents with N-acetylgalactosamine ligands conjugated in vivo.

The Lp(a) RNAi agent bound to the targeting ligand of Structure 101 was combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

The RNAi agent Lp(a), bound to a targeting ligand at the 5′ end of the sense strand, was delivered by SC injection. On day 1, 200 μl/20 g of mouse body weight of a solution of either saline or a dose of 1 mg/kg (mpk) of the RNAi agent AD03272 in buffered saline was injected subcutaneously into the fold of skin on the back between the shoulder blades. Four (4) Lp(a) Tg mice were used per treatment group.

Serum samples from treated mice were collected on days -1 (pre-dosing), 8, 15, 22, 29, 36 and 43. Knockdown was determined by calculating serum levels of circulating Lp(a) particles. Lp(a) particle levels were measured on a Cobas® Integra 400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by the pre-dosing expression level in that animal (in this case, the -1 day level) to determine the expression ratio normalized to -1 day. Expression at a specific time point was then normalized to the saline control group by dividing the -1 day normalized coefficient for an individual animal by the average of the -1 day normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. The experimental error is presented as standard deviation.

The results are shown in Fig. 15. AD03272 observed 88% knockdown at trough values (day 15) and maintained 75% knockdown at day 29. These data confirm that the targeting ligand of Structure 1008 can target LPA-targeted RNAi agents to the liver and provide >85% knockdown at a single dose administration of a dose of 1 mg/kg in transgenic mice.

Example 10. Knockdown of apolipoprotein(a) (apo(a)) in apo(a) transgenic (Tg) mice after administration of LP(a) expression-inhibiting oligomeric compounds (double-stranded RNAi agents) bound to a targeting ligand Structures 101, 102 and 103.

Oligomeric compounds inhibiting LP(a) expression (double-stranded Lp(a) RNAi agents) were prepared with the sequences presented in Table 1 below. 4.

- 42 045605

Table 6. LP(a) expression inhibitory oligomeric compounds (RNAi agent duplexes) from Example 10

Duplex ID: AD03275 5' Ά 3' SEQ ID NO: Sequence of the semantic chain: (AM04138-SS) (NAG25)uauausasguuaucgAfGfGfcucauucuc(invdA) 16 Antisense strand sequence: (AM04133-AS) usGfsagaauGfaGfccuCfgauaacucsusuau 17 Duplex ID: AD03341 5’ -» 3’ SEQ ID NO: Sequence of the semantic chain: (AM04233-SS) (NAG26)uauausasguuaucgAfGfGfcucauucuCM(invdA) 18 Antisense strand sequence: (AM04133-AS) usGfsagaauGfaGfccuCfgauaacucsusuau 19 Duplex ID: AD03421 5' 3' SEQ ID NO: Sequence of the semantic chain: (AM04372-SS) (NAG27)uauausasguuaucgAfGfGfcucauucuCM(invdA) 20 Antisense strand sequence: (AM04133-AS) usGfsagaauGfaGfccuCfgauaacucsusuau 21

In table 6 above the following notations are used:

In addition, (NAG25) is represented by the same structure as shown in Example 8 above and has a chemical structure that is represented herein by Structure 101. (NAG26) has a chemical structure that is represented herein by Structure 102. (NAG27) has a chemical structure, which is represented herein by Structure 103. As shown in the table above. 7, except for the choice of different targeting ligands, the compositions are identical.

Each strand of Lp(a) RNAi agents was synthesized according to solid-phase phosphoramidite technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to produce duplexes using methods generally described in Example 10 herein document.

Apo(a) transgenic (Tg) mice were used to evaluate the effectiveness of double-stranded RNAi agents with conjugated N-acetylgalactosamine ligands in vivo. Apo(a) Tg mice (Frazer KA et al 1995, Nature Genetics 9:424-431) (hereinafter referred to as apo(a) Tg mice) express human apo(a) with YAC containing the complete gene LPA (encoding apo(a) protein), with additional sequences in both the 5' and 3' directions.

Lp(a) RNAi agents bound to appropriate GalNAc ligands (i.e. (NAG25), (NAG26) or (NAG27)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art technology.

Lp(a) RNAi agents bound to the appropriate GalNAc ligands (i.e. (NAG25), (NAG26) or (NAG27)) at the 5′ end of the sense strand were delivered by SC injection. On day 1, 200 μl/20 g of mouse body weight of a solution containing either saline or a dose of 1 mg/kg (mpk) of the appropriate RNAi agent (AD03275, AD03341 or AD03421) was injected subcutaneously into the fold of skin on the back between the shoulder blades. in buffered saline solution. Three (3) apo(a) Tg mice were used per treatment group.

Serum samples from treated mice were collected on days -1 (pre-dosing), 8, 15, 22, 29, 36 and 43. Knockdown was determined by monitoring circulating serum apo(a) protein levels using the apo(a) ELISA (Abcam). For normalization, the apo(a) level for each animal at a given time point was divided by the pre-treatment expression level of that animal (in this case, the level for -1 day) to determine the expression ratio normalized to -1 day. Expression at a specific time point was then normalized to the saline control group by dividing the -1 day normalized coefficient for an individual animal by the average of the -1 day normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. The experimental error is presented as the standard error of the mean.

The results are shown in Fig. 16. For the Lp(a) RNAi agent AD03275 containing the targeting ligand Structure 101 (NAG25), 82% knockdown was observed at trough values (day 22) and maintained at 72% knockdown at day 29. For the Lp(a) RNAi agent AD03341, containing the targeting ligand Structure 102 (NAG26), 87% knockdown was observed at trough values (day 15), but the knockdown rate at day 29 was 45%, indicating an accelerated return to apo(a) levels. a) before dosing. For the Lp(a) RNAi agent AD03421, containing the targeting ligand Structure 103 (NAG27), 70% knockdown was observed at trough values (day 15); the knockdown rate on day 29 was 50%. These data confirm that all structures: Structure 101 (NAG25), Structure 102 (NAG26) and Structure 103 (NAG27), initially show similar knockdown activity. However, these data also show that AD03275 (Structure 101 (NAG25)) provides superior knockdown duration and maintenance (72% knockdown at day 29) compared to Structure 102 (NAG26) and Structure 103 (NAG27).

Example 11: LP(a) Expression Inhibitory Oligomeric Compounds (Double Stranded RNAi Agents) Bound to Structure 101 Targeting Ligand in Cynomolgus Macaques

Five different LPA RNAi agents bound to the targeting ligand represented by structure 101 were prepared to evaluate their efficacy in the cynomolgus monkey (Macaca fascicularis): AD03460, AD03536, AD03851, AD03853 and AD04110.

Each strand of Lp(a) RNAi agents was synthesized according to solid-phase phosphoramidite technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 4 herein.

Targeting ligands for all five (5) Lp(a) RNAi agents were added to the 5' end of the sense strand using non-nucleoside phosphoramidite synthesis generally described herein and known in the art. The targeting ligand for each of the Lp(a) RNAi agents was coupled to the 5' end of the corresponding RNAi agent using the following phosphoramidite compound:

- 44 045605

AD03460 and AD03536 included a targeting ligand (NAG25) conjugated to the 5′ end of the sense strand of the corresponding RNAi agent. (NAG25) had the same structure as shown in Example 8 above.

AD03851, AD03853 and AD04110 contained a targeting ligand (NAG25)s conjugated to the 5' end of the sense strand of the corresponding RNAi agent.

Blood samples were collected and analyzed for lipoprotein(a) levels on days 8 and 15. Lp(a) levels were normalized to the average of three pre-dosing values. Normalized Lp(a) levels are shown in the table below:

Normalized Lp(a), day 8 Normalized Lp(a), 15 day Saline solution 1.01 ±0.06 1.15 ±0.07 AD03460 0.68 ±0.12 0.40 ±0.13 ADO3536 0.54 ±0.07 0.21 ±0.06 ADO3851 0.41 ±0.08 0.18 ±0.08 ADO3853 0.50 ±0.23 0.27 ±0.17 AD04110 0.59 ±0.13 0.43 ±0.10

These data show that when administered at 2 mg/kg (mpk) doses of several different Lp(a) RNAi agents conjugated to the same targeting ligand structure, Structure 101, presented herein, significant knockdown was achieved in cynomolgus monkeys.

Example 12: F12 expression inhibitory oligomeric compounds (double-stranded RNAi agents) bound to Structure 101 targeting ligands in cynomolgus monkeys.

Oligomeric compounds inhibiting F12 expression (double-stranded F12 RNAi agents) were prepared with the sequences presented in Table 1 below. 7.

Table 7. F12 expression inhibitory oligomeric compounds (RNAi agent duplexes) from Example 12

Duplex ID: AD03635 5’ -» 3’ SEQ ID NO: Sequence of the semantic chain: (AM04130-SS) (NAG25)uauaugscsccaagaAfaGfugaaagacc(invdA) 22 Antisense strand sequence: (AM03157-AS) usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 23

In table 7 above (NAG25) corresponds to the same structure as shown in Example 8 above and is represented by Structure 101 herein.

Each strand of Lp(a) RNAi agents was synthesized according to solid-phase phosphoramidite technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to produce duplexes using methods generally described in Example 4 herein document.

An F12 RNAi agent conjugated to a targeting ligand at the 5' end of the sense strand was prepared and combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

On day 1, cynomolgus monkeys (Macaca fascicularis) were injected subcutaneously with 3 mg/kg AD03635. Doses were administered to three (3) monkeys per treatment group.

Serum samples from treated cynomolgus monkeys were collected on days -7 and 1 (pre-dosing); and on days 8, 15, and 22 to monitor knockdown. Knockdown was measured by quantifying circulating cynomolgus F12 protein (cF12) levels in serum using a human F12 ELISA kit (Molecular Innovations). The cF12 levels for each animal at the corresponding time point were divided by the pre-treatment expression level of the indicated animal (average of day -7 and day 1) to determine the expression ratio normalized to the pre-dosing level. The experimental error is presented as standard deviation.

In fig. Figure 17 shows the results. Knockdown was observed when the RNAi agent F12 associated with (NAG25) was administered to cynomolgus monkeys (Structure 101 herein).

Example 13: Alpha-1-antitrypsin expression-inhibiting oligomeric compounds (double-stranded RNAi agents) bound to Structure 101 targeting ligands in PiZ transgenic mice.

The PiZ transgenic mouse model (PiZ mice) was used to evaluate in vivo RNAi agents targeting the alpha-1 antitrypsin (AAT) gene. PiZ mice are carriers of the mutant human AAT allele PiZ and a model of human alpha-1 antitrypsin deficiency (AATD) (Carlson et al., Journal of Clinical Investigation 1989).

Oligomeric compounds (double-stranded RNAi agents) inhibiting AAT expression were obtained with the sequences presented below in the table. 8.

Table 8. Oligomeric compounds inhibiting AAT expression (RNAi agent duplexes) from example 13

Duplex ID: AD04454 5' A 3' SEQ ID NO: Sequence of the semantic chain: (AM05662-SS) (NAG25)scsgauaucaUfCfAfccaaguuccsa(invAb) 24 Antisense strand sequence: (AM05663-AS) usGfsgAfaCfuugguGfaUfgAfuAfusCfsg 25

In table 8 (NAG25)s has the chemical structure shown in Example 11 above.

The RNAi agent AAT was prepared in a pharmaceutically acceptable saline buffer and injected subcutaneously (s.c.) into the fold of skin on the back between the shoulder blades of PiZ mice with 200 μl of solution/20 g of mouse body weight to evaluate the knockdown of AAT gene expression. Each mouse received one SC dose of 5 mg/kg (mpk) AD04454. Doses of the RNAi agent AAT were administered to three mice (n=3).

Plasma samples were collected and analyzed for Z-AAT protein levels on days -1, 1 (pre-dosing), 8 and 15. AAT levels were normalized to plasma AAT levels on day 1 (pre-dosing). Protein levels were measured by quantifying circulating levels of human plasma Z-AAT using an ELISA kit.

Mean normalized AAT levels (Z-AAT) are shown in FIG. 18. Knockdown was observed using the RNAi agent AAT bound to the targeting ligand Structure 101 presented herein in PiZ transgenic mice.

Example 14 Knockdown of F12 following administration of F12 expression inhibitory oligomeric compounds (double-stranded RNAi agents) bound to Structure 101 targeting ligands in wild-type mice

Oligomeric compounds inhibiting F12 expression (double-stranded F12 RNAi agents) were prepared with the sequences presented in Table 1 below. 9.

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Claims (29)

Table 9. F12 expression inhibitory oligomeric compounds (RNAi agent duplexes) from Example 14. Duplex ID: AD03632 5’ -» 3’ SEQ ID NO: Sequence of the semantic chain: (AM04613-SS) (NAG25)gcgaugscsccaagaAfaGfugaaagacc(invdA) 26 Antisense strand sequence: (AM04048-AS) usGfsgucuuUfcAfcuuUfcuugggcsasucgc 27 In table 9 (NAG25) has the chemical structure shown in Example 8 above and is represented by Structure 101 as described herein. Each strand of F12 RNAi agents was synthesized using solid-phase phosphoramidite technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein. F12 RNAi agents conjugated to the appropriate GalNAc targeting ligand (ie (NAG25)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art. This composition was delivered by subcutaneous injection. On day 1, 200 μl/20 g of mouse body weight of a solution containing either saline or a dose of 3 mg/kg (mpk) AD03632 in buffered saline was injected subcutaneously into the fold of skin on the back between the shoulder blades. Three (3) wild-type mice were used per treatment group. As shown above, AD03632 includes a structure (NAG25) attached at the 5' end of the sense strand. Serum samples from treated mice were collected on days -1 (pre-dosing), 8, 15, 22, 29 and 36 to monitor knockdown. Knockdown was measured by quantifying circulating levels of mouse F12 protein (mF12) in serum using an in-house mF12 assay developed based on alphaLISA® (Perkin Elmer). mF12 levels for each animal at the corresponding time point were divided by the pre-treatment expression level in that animal to determine the expression ratio normalized to the pre-dosing level. Expression at a specific time point was then normalized to the saline control group by dividing the pre-dose day-normalized coefficient for an individual animal by the average pre-dose day-normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. The experimental error is presented as standard deviation. The results of this study are shown in Fig. 19. When using AD03632, which includes the targeting ligand of Structure 101 as described herein, significant knockdown was observed at all time points. Other implementation options It should be understood that although the present invention has been described in conjunction with a detailed description thereof, the above description is intended to be illustrative and not to limit the scope of the invention, which is defined by the scope of the appended claims. The scope of the following claims includes other aspects, advantages and modifications. CLAIM 1. A targeting ligand for directing a therapeutic compound to a target in vivo or in vitro, containing the structure of formula B TARGETTING _o FRAGMENT \X\qX\/ TARGETTING FRAGMENT Π ⁰ G TARGETTING _χ ___ _NH JL FRAGMENT - (/NX Er NH O (Formula B), - 47 045605 or a pharmaceutically acceptable salt thereof, where n is an integer from 1 to 20; X represents O, S or NH and said targeting moiety is selected from the group consisting of N-acetylgalactosamine, galactose, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine and N-isobutanoylgalactosamine. 2. Targeting ligand according to claim 1, containing the structure where n is an integer from 1 to 20, or a pharmaceutically acceptable salt thereof. 3. The targeting ligand according to claim 1, where the targeting ligand contains a structure selected from or a pharmaceutically acceptable salt of any of structures 101, 102 or 103. 4. The targeting ligand according to claim 1, where the targeting moiety is or a pharmaceutically acceptable salt thereof. 5. Targeting ligand according to claims 1, 2 or 4, where n is 6. 6. Targeting ligand according to claims 1, 2 or 4, where n is 8. 7. The targeting ligand according to any one of claims 1 to 6, wherein the targeting ligand is associated with an RNAi agent. 8. The targeting ligand according to claim 7, wherein the RNAi agent is double-stranded. 9. The targeting ligand of claim 8, wherein the RNAi agent comprises one or more modified nucleotides. 10. The targeting ligand according to any one of claims 1 to 9, additionally containing an RNAi agent bound - 48 045605 with a targeting ligand at the 3’ or 5’ end of the RNAi agent. 11. The targeting ligand according to claim 10, wherein the RNAi agent is double-stranded. 12. The targeting ligand according to claim 11, wherein the double-stranded RNAi agent is linked to the targeting ligand 5' end of the sense strand of the RNAi agent. 13. The targeting ligand of claim 12, wherein the RNAi agent is linked to the targeting ligand via a phosphate group, a phosphorothioate group, or a phosphonate group. 14. A pharmaceutical composition containing the targeting ligand according to claim 1, where the binding ligand is associated with an RNAi agent, and the structure of the targeting ligand and the RNAi agent is represented by a structure selected from the group consisting of where Z includes or consists of an RNAi agent; where Z includes or consists of an RNAi agent; And where Z includes or consists of an RNAi agent, or a pharmaceutically acceptable salt of any of structures 101a, 102a or 103a. 15. The pharmaceutical composition according to claim 14, wherein the RNAi agent is a double-stranded RNAi agent. 16. The pharmaceutical composition according to claim 15, wherein the double-stranded RNAi agent is linked to a targeting ligand at the 5' end of the sense strand of the RNAi agent. 17. A phosphoroamidite compound containing a targeting ligand for directing a therapeutic compound to a target in vivo or in vitro, wherein said phosphoroamidite compound has the structure where n is an integer from 1 to 20, or a pharmaceutically acceptable salt thereof. 18. A compound according to claim 17, having a structure selected from the group consisting of - 49 045605 (Structure 103 d) or a pharmaceutically acceptable salt of any of structures 101d, 102d or 103 d. 19. A compound according to claim 18, wherein said compound is or a pharmaceutically acceptable salt thereof. 20. A method of inhibiting the expression of a target nucleic acid in a subject, comprising administering a therapeutic amount of an RNAi agent conjugated to any of the targeting ligands of claims 1 to 13 or a pharmaceutically acceptable salt thereof. 21. A method of introducing an RNAi agent into a mammalian cell, comprising contacting the mammalian cell with a targeting ligand according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof associated with the RNAi agent. 22. The method according to claim 21, characterized in that the specified cell is located in the body of the subject. 23. The method according to claim 22, characterized in that the subject is a human. 24. A method of treating a disease or disorder that would benefit from reducing the level of a target mRNA or inhibiting the expression of a target gene, comprising administering a therapeutic amount of a targeting ligand according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof coupled to an RNAi agent, subject in need of such treatment. 25. A method of treating a disease or disorder that would benefit from administration of an RNAi agent, comprising administering a therapeutic amount of a composition according to any one of claims 14 to 16 to a subject in need of such treatment. 26. A compound containing a targeting ligand associated with an RNAi agent, where the specified compound is selected from the group consisting of - 50 045605 where n is an integer from 1 to 20 and Z contains the RNAi agent; And where n is an integer from 1 to 20, and Z contains an RNAi agent, or a pharmaceutically acceptable salt of any of these structures. 27. A compound according to claim 26 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of where Z contains the RNAi agent; where Z contains the RNAi agent; And where Z contains an RNAi agent, or a pharmaceutically acceptable salt of any of these structures. 28. A compound according to claim 26 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of - 51 045605 where Z contains the RNAi agent; And (Structure 103 s). where Z contains an RNAi agent, or a pharmaceutically acceptable salt of any of these structures. 29. A targeting ligand for directing a therapeutic compound to a target in vivo or in vitro, comprising a structure selected from the group consisting of or a pharmaceutically acceptable salt of any of NAG25, NAG26 or NAG27.